PSMA TARGETING UREA‐BASED LIGANDS FOR PROSTATE CANCE R RADIOTHERAPY AND  IMAGING    Field of the Invention  The present invention relates to urea‐based ligands� �specifically targeting a prostate‐specific  membrane antigen and their use in radiotherapy and i maging.  Background of the invention  Prostate cancer (PC) is one of the most commonly di agnosed diseases in men. ¹  Moreover, a  significant amount of people suffering from PC will  develop bone metastases, which results in  a 1‐year survival rate of only 40 %. Some of the  patients are non‐responders to conventional  hormonal therapy, developing what is known as castrat ion‐resistant prostate cancer (CRPC). ^2,3   Limited options are available for patients suffering  from CRPC, for which reason the  development of highly specific and potent radiopharmac euticals is of the utmost interest.  Targeting prostate‐specific membrane antigen (PSMA) w hich is expressed 8‐to‐12‐fold higher  in PC cells when compared to healthy tissue, ⁴  offers the possibility for bio‐specific imagi ng and  treatment of PC. Nevertheless, some of the developed� �PSMA‐targeting radiopharmaceuticals  have significantly unpleasant side effects, like renal  toxicity and salivary gland build‐up.  The use of small molecular weight ligands for select ively targeting PSMA stands to reason, as it  fulfils all the requirements for radiotherapy. Small  molecules exhibit the best pharmacokinetic  properties such as short half‐life in the bloodstre am and fast clearance. In contrast, big  biological entities such as antibodies have much long er circulation times and a very slow  clearance profile. Long circulation time and a slow  clearance profile makes a compound less  suitable for radiotherapy, since the extended presence  of the noxious radioactive payload in  the bloodstream translates into unwanted damage in no n‐target tissue. In the small‐molecule  class, urea‐based ligands have been shown to exhibi t high affinities for PSMA, with very low  non‐specific binding, high tumour accumulation over  time and fast clearance.   The current state‐of‐the‐art in PSMA targeted ra diotherapy is based on  ¹⁷⁷ Lu‐PSMA‐617, which  has shown good molecular response in clinical evaluat ions, clearing a noticeable amount of  metastases and significantly reducing PSA concentration s to normal levels (below 4.0 ng/mL). ⁵   Nevertheless, a notable 30% of patients do not respo nd to β ^‐ ‐emitter based therapy such as  ¹⁷⁷ Lu‐PSMA‐617, for which reason an alternative� �strategy is to use cytotoxic alpha‐emitters  instead.  Efforts have been made in targeting PSMA for therapy  employing alpha‐emitters. For example,  ²²⁵ Ac has been used but it is not an ideal radi onuclide for therapy as it decays through a chain  that includes four α‐active daughter radionuclides.� �Combined with a decay half‐life of 10 days,  this means that extensive damage can be caused to h ealthy tissue once the radionuclides are  expelled from the chelator. Our approach is based on  the use of Astatine‐211 ( ²¹¹ At). Astatine‐ 211 is one of the most appealing radionuclides for  alpha‐radiotherapy. Its short half‐life of 7.2  hours is in accordance with the pharmacokinetics of  small‐molecule urea‐based PSMA ligands.  Moreover, its decay pathways do not include any long ‐lived alpha‐emitting daughter that  could be expelled into the bloodstream from the bind ing site. This significantly reduces any  unwanted cytotoxicity to the patient.   Radiohalogenated PSMA‐targeting pharmaceuticals have b een developed and have shown  good, specific tumour uptake, but these compounds wer e marred by renal and salivary gland  build‐up, when no blocking agents were administered. ⁶    Herein, we describe PSMA‐targeting radiopharmaceutical s labeled with one or more nuclides  or radionuclides of the halogen group, applicable for  imaging, radiotherapy or theranostics,  depending on the specific radionuclide or combination� �of radionuclides selected.   The radionuclide  ²¹¹ At is a therapeutic radionuclide that emits alp ha particles. Alpha particles  have particular properties that set them apart from  other types of therapeutic radionuclides.  Notably, alpha particles differ from beta particles,  such as emitted by lutetium‐177 ( ¹⁷⁷ Lu),  iodine‐131 ( ¹³¹ I) or yttrium‐90 ( ⁹⁰ Y), by having substantially shorter range in ti ssue and by  depositing a higher level of energy along their path . Further, alpha particles travel by straight  paths, whereas beta particles travel by tortuous path s, and alpha particle energy deposition is  characterized by a Bragg peak. The shorter range of� �alpha particles make them more effective  against micrometastases, as the energy is linearly de posited with a range of less than about 10  cancer cells. Further, the high energy deposition of� �alpha particles make direct double  stranded DNA breaks more likely, with these having a  higher chance of killing the cancer cell  due to the difficulty of repair. Beta particles have  less dense energy deposition, resulting in  DNA damage occurring indirectly through the generation  of reactive oxygen species (ROS) and  being single‐stranded in nature. These features make  alpha particle emitters more damaging  than beta emitters on a decay‐by‐decay basis.   Accordingly, head‐to‐head comparisons between alpha� �and beta emitters are not easily  designed nor evaluated, as the two modalities have d ifferent responses in different tumor  models, and they are employed at different radioactiv ity levels. In addition, the available   

relevant alpha emitters (Pb‐212, Ac‐225, Th‐22 7 and At‐211) also have vastly different  properties, notably decay half‐lives and decay chain s, making each radionuclide having unique  cytotoxicity and side‐effect profiles.   The current state‐of‐the‐art therapeutic variant  in clinical use for beta‐particle therapy is  177Lu‐PSMA‐617, while a variant for alpha‐particl e radiotherapy labeled with actinium‐225 is  also reported ¹⁰ . For theranostic imaging, gallium‐68 is most� �commonly used. These compounds  are radiolabeled in a DOTA chelator situated at the� �distal end of the molecule, which enables  labeling with radiometals, such as Ga and Lu. Howeve r, using a chelator such as the DOTA  chelator for radiolabeling is only an option in rela tion to radiometals and accordingly, since  astatine‐211 is not a radiometal but a halogen, a� �different strategy must be used for providing  At‐211 radiolabeled PSMA.   Herein, we have provided a compound series where the  halogen nuclide or radionuclide, such  as astatine‐211, is placed in the aromatic linker  region, providing a drastic difference in  structure and radiolabeling technique from the compoun ds using radiometals. We here report  that such radiochemical modifications of the linker r egion are possible without compromising  cellular internalization, something which is considered  a prerequisite for therapeutic success.  Despite radioastatination of the linker region, intern alization that is on par than PSMA‐617 can  be obtained, depending on the specific position of t he astatine‐211.   For compounds labeled with astatine‐211 in this way , theranostic companions for imaging are  highly relevant, such is analogues that are structura lly identical, but with the radionuclide  exchanged for e.g. fluorine‐18, iodine‐123, iodine� ��125, iodine‐131 or iodine‐124. These  radionuclides are also halogens, like astatine‐211 a nd are therefore well‐suited for preparing  theranostic companions, labeled in the same position  in the linker, using related aromatic  substitution radiochemistry. As per their close struct ural similarity, such compounds are  expected to have advantages mirroring those demonstrat ed for the astatine‐211 labeled  compounds.  We have found that the compounds disclosed herein ca n be labeled with astatine‐211 in a  markedly higher radiochemical yield than reported anal ogous compounds, which is a  substantial advantage. We believe that this may be d ue to the differences in molecular  structure between our compounds and previously describ ed compounds, although there is  currently no reported theoretical basis for why this� �occurs.   Other radiopharmaceuticals developed (WO2019157037A1) we re urea‐based and DOTA  containing, but also contained an aliphatic chain as� �well as a tertiary amide as key features.   

Tertiary amides are prone to hydrolysis in vivo ^11, 12 . The cyclohexyl group featured in our  compounds has been shown to favour internalisation an d thus, tumour accumulation. ^5,7  High  cellular internalization is regarded as a favorable p roperty in PSMA‐targeted radiotherapy.  The PSMA targeting urea‐based ligands for prostate  cancer radiotherapy and imaging disclosed  herein are based on peptide bonds. Thus, they do no t bear any tertiary amides as the  radiohalogen bearing moiety and are more stable under  physiological conditions. The ease of  synthesis of amide bonds through chemically modified  amino acids makes these PSMA  targeting urea‐based ligands for prostate cancer rad iotherapy and imaging easy to  manufacture in an automated, resin‐bound or solid‐ phase process if needed to scale up for  routine clinical use.   The compounds disclosed herein showed excellent cellul ar internalization profiles. Cellular  internalization data have not previously been reported  for PSMA compounds modified with  astatine‐211 in this region of the molecule.  It  is therefore surprising that even with synthetic  modification of the linker amino acid structure, high  internalization can still be achieved, on  par than optimized compounds clinically used in β ^‐ ‐particle radiotherapy (such as  ¹⁷⁷ Lu‐PSMA‐ 617).   Summary of the invention  The present invention provides novel PSMA targeting u rea‐based ligands and the use of these  compounds in radiotherapy and imaging is disclosed. � � The PSMA targeting urea‐based ligands of the presen t invention have the following general  formula (I):    wherein:   A is independently carboxylic acid, sulphonic acid, p hosponic acid, tetrazole or isoxazole;   L is selected from the group consisting of urea, th iourea, ‐NH‐(C=O)‐O‐, ‐O‐(C=O)‐NH‐ or � �CH ₂ ‐ (C=O)‐CH ₂ ‐ ,    K is selected from the group consisting of –(C=O)� ��NH‐, ‐CH ₂ ‐NH‐(C=O)‐ or  wherein  p is independently an integer selected from the grou p consisting of 1, 2, 3, 4, 5 and 6;   q is an integer selected from the group consisting  of 0, 1, 2, 3, 4, 5 and 6;  Y is selected from the group consisting of:  wherein  Q ₁  is –C–R ₃  or N, wherein R ₃  is H or C ₁ ‐C ₅  alkyl;   Q ₂  is O, S or NH;   Hal is a nuclide or radionuclide of the halogen gro up selected from the group consisting of  isotopes and radioisotopes of fluorine, iodine, bromin e or astatine;   M is a chelating agent, that can comprise a metal  n is an integer selected from the group consisting  of 1, 2, 3, 4, 5 and 6;   m is an integer selected from the group consisting  of 0 and 1;   o is an integer selected from the group consisting  of 0 and 1;   R ₁  is –CH–CH ₂ –Z or  –CH–CH ₂ –Y;  wherein Z is selected from the group consisting of:� � and Y is selected from the group consisting of:        wherein  Q ₁  is –C–R ³  or N, wherein R ³  is H or C ₁ ‐C ₅  alkyl;   Q ₂  is O, S or NH;   Hal is a nuclide or radionuclide of the halogen gro up selected from the group consisting of  isotopes and radioisotopes of fluorine, iodine, bromin e or astatine;   R ₂  is –CH–CH ₂ –Y or  –CH ₂ –X–;   wherein X is an aromatic monocyclic or polycyclic ri ng system having 6 to 14 carbon atoms,  cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloh eptyl or cyclooctyl;  and Y is selected from the group consisting of:  wherein  Q ₁  is –C–R ³  or N, wherein R ³  is H or C ₁ ‐C ₅  alkyl;   Q ₂  is O, S or NH;   Hal is a nuclide or radionuclide of the halogen gro up selected from the group consisting of  isotopes and radioisotopes of fluorine, iodine, bromin e or astatine;   and wherein formula (I) comprises at least one isoto pe or radioisotope selected from fluorine,  iodine, bromine or astatine;  and pharmaceutically acceptable salts thereof.  Moreover, the invention relates to compounds of the  general formula (I) but having a non‐ radioactive isotope of fluorine, iodine or bromine in stead of a radioisotope of fluorine, iodine,  bromine or astatine.   The suitability of the compounds of Formula (I) in  radiotherapy and imaging is shown.      

Brief description of the drawings  Figure 1 shows the structural formula (I) and (Ia)  of the compounds of the present invention.  Figure 2A shows the time activity curve of non‐tar get tissue for compound PSMA‐617.  Figure 2B shows the time activity curve of tumor/mus cle for compound PSMA‐617.  Figure 3A shows the time activity curve of non‐tar get tissue for compound Ii.  Figure 3B shows the time activity curve of tumor/mus cle for compound Ii.  Figure 4A shows the time activity curve of non‐tar get tissue for compound Ij.  Figure 4B shows the time activity curve of tumor/mus cle for compound Ij.  Figure 5A shows the time activity curve of non‐tar get tissue for compound Ik.  Figure 5B shows the time activity curve of tumor/mus cle for compound Ik.  Figure 6A shows the time activity curve of non‐tar get tissue for compound Il.  Figure 6B shows the time activity curve of tumor/mus cle for compound Il.  Figure 7A shows the time activity curve of non‐tar get tissue for compound Im.  Figure 7B shows the time activity curve of tumor/mus cle for compound Im.    Detailed description of the invention  The PSMA targeting urea‐based ligands of the presen t invention are suitable for use as  radiopharmaceuticals, either as imaging agents or for� �the treatment of prostate cancer, or as  theranostic agents.  The PSMA targeting urea‐based ligands of the presen t invention take advantage of a urea‐ based binding motif (((S)‐5‐amino‐1‐carboxypentyl )carbamoyl)‐L‐glutamic acid). This motif  specifically interacts with the PSMA antigen binding  pocket. It contains an urea that forms a  coordination complex with a Zn ⁺²  atom, which is crucial for the binding. Moreo ver, carboxylic  acids also interact with the residues in the vicinit y of the binding site, making this scaffold very  convenient for PSMA‐specific targeting.  The compounds disclosed herein comprises at least one  isotope or radioisotope selected from  the halogen group and are suitable for different pur poses. The halogen astatine, particularly  the radioactive radionuclide  ²¹¹ At, is particularly useful in alpha‐particle t herapy, whereas  ¹⁸ F   

and the radionuclides of iodine  ¹²⁵ I,  ¹²³ I,  ¹³¹ I, and  ¹²⁴ I, are primarily intended as theranostic  companions for the astatine‐211 labeled variant. In� �this sense,  ¹⁸ F and most radioisotopes of  iodine are suitable for imaging. Presently, the appro ach is that patients be first diagnosed  using diagnostic imaging, for example with a compound  labeled with  ¹⁸ F or radioiodine, and  then treated with a modality such as alpha‐particle  therapy. For this, analogues labeled with  radionuclides for imaging are therefore required. For� �theranostics to work best, the diagnostic  variant must have the radionuclide placed in the exa ct same position as the therapeutic  variant, and the rest of the molecule should be ide ntical.  The bromine radionuclides  ⁷⁷ Br and  ⁸⁰ Br are primarily relevant for Auger electron ra diotherapy  and  ¹²⁵ I and  ¹²³ I have also been used for such a purpose. Aug er therapy is a form of radiation  therapy for the treatment of cancer which relies on� �a large number of low‐energy electrons  (emitted by the Auger effect) to damage cancer cells , rather than the high‐energy radiation  used in traditional radiation therapy.  Similar to o ther forms of radiation therapy, Auger  therapy relies on radiation‐induced damage to cancer  cells (particularly DNA damage) to arrest  cell division, stop tumor growth and metastasis and  kill cancerous cells. It differs from other  types of radiation therapy in that electrons emitted� �via the Auger electrons are released in  large numbers with low kinetic energy.  Non‐radioactive test‐compounds corresponding to the� �radioactive compounds but comprising  a non‐radioactive isotope of iodine, fluorine and b romine, respectively, can be applied instead  of the radioactive variants in order to test the ap plicability of the compounds. Such test‐ compounds preferably comprises one of the non‐radioa ctive isotopes  ¹²⁷ I,  ¹⁹ F,  ⁷⁹ Br or  ⁸¹ Br,  respectively, instead of the radioactive variants. The re is, however, no non‐radioactive isotope  for astatine, but  ¹²⁷ I can be used as a test‐compound instead. Fo r instance, in order to test the  impact of influencing the linker region, iodine  ¹²⁷ I isotope were provided and tested herein.  ¹²⁷ I  is a large atom (atomic radius: 198 pm) and similar  in size to astatine‐211 (atomic radius: 200  pm) and with a highly similar halogen electronic con figuration. Accordingly, experiments using  the non‐radioactive  ¹²⁷ I‐compound was applied to demonstrate that the  linker region can be  modified with a large halogen and still display effi cient internalization, on par with or better  than reported, optimized compounds in clinical use.  Surprisingly, despite the synthetic modifications in k ey regions, the binding profile and  biodistribution/pharmacokinetics of various of the here in disclosed new compounds are  comparable or superior to values observed for PSMA‐ 617 in direct head‐to‐head comparison.    This is particularly true for compounds Ii and Im.  PSMA‐617 is the most clinically applied  therapeutic PSMA inhibitor.   The term “nuclide” comprises both non‐radioactive  and radioactive nuclides (radionuclides).  The compounds of the present invention can comprise  either a radioactive or non‐radioactive  nuclide depending on the intended use. When used her ein in relation to specific compounds  the terms “nuclide” and “radionuclide” are use d to make an indication of whether the final  compound is radioactive or not.   The term “isotope” comprises both non‐radioactive  and radioactive isotopes (radioisotopes).  The compounds of the present invention can comprise  either a radioactive or non‐radioactive  isotope depending on the intended use. When used her ein in relation to specific compounds  the terms “isotope” and “radioisotope” are use d to make an indication of whether the final  compound is radioactive or not. The various isotopes� �of the halogen nuclides iodine, fluorine,  bromine and astatine are all well known, and the is otope number will reveal whether the  isotope is stable (non‐radioactive) or not (radioact ive).  The PSMA targeting urea‐based ligands of the presen t invention have the following general  formula (I):  wherein:   A is independently carboxylic acid, sulphonic acid, p hosponic acid, tetrazole or isoxazole;   L is selected from the group consisting of urea, th iourea, ‐NH‐(C=O)‐O‐, ‐O‐(C=O)‐NH‐ or � �CH ₂ ‐ (C=O)‐CH ₂ ‐;  K is selected from the group consisting of –(C=O)� ��NH‐, ‐CH ₂ ‐NH‐(C=O)‐ or  wherein  p is independently an integer selected from the grou p consisting of 1, 2, 3, 4, 5 and 6;     q is an integer selected from the group consisting  of 0, 1, 2, 3, 4, 5 and 6;  Y is selected from the group consisting of:  wherein  Q ₁  is –C–R ³  or N, wherein R ³  is H or C ₁ ‐C ₅  alkyl;   Q ₂  is O, S or NH;   Hal is a nuclide or radionuclide of the halogen gro up selected from the group consisting of  isotopes and radioisotopes of fluorine, iodine, bromin e or astatine; M is a chelating agent, that  can comprise a metal,  n is an integer selected from the group consisting  of 1, 2, 3, 4, 5 and 6;   m is an integer selected from the group consisting  of 0 and 1;   o is an integer selected from the group consisting  of 0 and 1;   R ₁  is –CH–CH ₂ –Z or  –CH–CH ₂ –Y;  wherein Z is selected from the group consisting of:� � and Y is selected from the group consisting of:  wherein  Q ₁  is –C–R ³  or N, wherein R ³  is H or C ₁ ‐C ₅  alkyl;   Q ₂  is O, S or NH;   Hal is a nuclide or radionuclide of the halogen gro up selected from the group consisting of  isotopes and radioisotopes of fluorine, iodine, bromin e or astatine;     R ₂  is –CH–CH ₂ –Y or  –CH ₂ –X–;   wherein X is an aromatic monocyclic or polycyclic ri ng system having 6 to 14 carbon atoms,  cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloh eptyl or cyclooctyl;  and Y is selected from the group consisting of:  wherein  Q ₁  is –C–R ³  or N, wherein R ³  is H or C ₁ ‐C ₅  alkyl;   Q ₂  is O, S or NH;   Hal is a nuclide or radionuclide of the halogen gro up selected from the group consisting of  isotopes and radioisotopes of fluorine, iodine, bromin e or astatine;   and wherein formula (I) comprises at least one isoto pe or radioisotope selected from fluorine,  iodine, bromine or astatine,  and pharmaceutically acceptable salts thereof.  In a particular embodiment, the PSMA targeting ligand  of formula (I) is selected from the  group of compounds of formula (Ia):  wherein:   A is independently carboxylic acid, sulphonic acid, p hosponic acid, tetrazole or isoxazole;   n is an integer selected from the group consisting  of 1, 2, 3  and 4 ;   m is an integer selected from the group consisting  of 0 and 1;   o is an integer selected from the group consisting  of 0 and 1;   Y is selected from the group consisting of:      wherein  Q ₁  is –C–R ³  or N, wherein R ³  is H or C ₁ ‐C ₅  alkyl;   Q ₂  is O, S or NH;   Hal is selected from the group consisting of isotope s and radioisotopes of fluorine, iodine,  bromine or astatine;   M is a chelating agent that can comprise a metal  R ₁  is –CH–CH ₂ –Z or  –CH–CH ₂ –Y;  wherein Z is selected from the group consisting of:� � and Y is selected from the group consisting of:    wherein  Q ₁  is –C–R ³  or N, wherein R ³  is H or C ₁ ‐C ₅  alkyl;   Q ₂  is O, S or NH;   Hal (halogen) is selected from the group consisting  of isotopes and radioisotopes of fluorine,  iodine, bromine or astatine;   R ₂  is –CH–CH ₂ –Y or  –CH ₂ –X–;   wherein X is an aromatic monocyclic or polycyclic ri ng system having 6 to 14 carbon atoms,  cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloh eptyl or cyclooctyl;  and Y is selected from the group consisting of:      wherein  Q ₁  is -C-R ³  or N, wherein R ³  is H or C ₁ ‐C ₅  alkyl;   Q ₂  is O, S or NH;   Hal is selected from the group consisting of isotope s and radioisotopes of fluorine, iodine,  bromine or astatine;   and wherein formula (I) comprises at least one isoto pe or radioisotope selected from fluorine,  iodine, bromine or astatine;  and pharmaceutically acceptable salts thereof.   The chelating agent may be selected from one of the  following chelators:   1,4,7,10‐tetraazacyclododecane‐N,N',N',N"‐tetraacetic� �acid (DOTA),   N,N'‐bis(2‐hydroxy‐5‐(carboxyethyl)benzyl)ethylenedi amine N,N'‐diacetic acid (HBED‐CC),   14,7‐triazacyclononane‐1,4,7‐triacetic acid (NOTA),� �  2‐(4.7‐bis(carboxymethyl)‐1,4,7‐triazonan‐1‐yl)p entanedioic acid (NODAGA),   2‐(4,7,10‐tris(carboxymethyl)‐1,4,7,10‐tetraazacyclo dodecan‐1‐ yl)pentanedioic acid  (DOTAGA),   14,7‐triazacyclononane phosphinic acid (TRAP),   14,7‐triazacyclononane‐1‐methyl(2‐carboxyethyl)phosp hinic acid‐4,7‐bis(methyl(2‐ hydroxymethyl)phosphinic acid (NOPO),   3,6,9,15‐tetraazabicyclo9.3.1.pentadeca‐1 (15),11,13‐ triene‐3,6,9‐ triacetic acid (PCTA),   N'‐(5‐acetyl (hydroxy)aminopentyl‐N‐(5‐(4‐(5‐  aminopentyl)(hydroxy)amino‐4‐ oxobutanoyl)amino)pentyl‐N‐ hydroxysuccinamide (DFO),� �  diethylenetriaminepentaacetic acid (DTPA),   trans‐cyclohexyl‐diethylenetriaminepentaacetic acid (C HX‐DTPA),   1‐oxa‐4,7,10‐triazacyclododecane‐4,7,10‐triacetic� �acid (OXO‐Do3A),   

p‐isothiocyanatobenzyl‐DTPA (SCN‐BZ‐DTPA),   1‐(p‐isothiocyanatobenzyl)‐3‐methyl‐DTPA (1B3M),� �  2‐(p‐isothiocyanatobenzyl)‐4‐methyl‐DTPA (1M3B),� �  1‐(2)‐methyl‐4‐isocyanatobenzyl‐DTPA (MX‐DTPA),  that can comprise a metal; and  pharmaceutically acceptable salts thereof.  In some embodiments, the chelating agent comprises a� �metal. In a preferred embodiment, the  chelating agent comprises a metal selected from the  group consisting of Y, Lu, Tc, Zr, In, Sm,  Re, Cu, Pb, Ac, Bi, Al, Ga, Ho and Sc.  The nuclide or radionuclide (Hal) may be present in� �the R ₁ , R ₂  and Y groups in Formula (I). The  nuclide or radionuclide is selected from the halogen� �group. This group comprises isotopes and  radioisotopes of Fluorine (F), Chlorine (Cl), Bromine� �(Br), Iodine (I) and Astatine (At).   In a preferred embodiment, the halogen nuclide is a� �radionuclide selected from a radioisotope  of fluorine, a radioisotope of iodine, a radioisotope  of bromine or a radioisotope of astatine.   In another preferred embodiment, the halogen nuclide  is a radionuclide selected from the  group consisting of  ¹⁸ F,  ¹²⁵ I,  ¹²³ I,  ¹³¹ I,  ¹²⁴ I,  ²¹¹ At,  ⁷⁷ Br and  ⁸⁰ Br.   In a particularly preferred embodiment, the halogen n uclide is one of the following  radionuclides  ^{123/124/125/131} I or  ²¹¹ At.  In another preferred embodiment, the nuclide is non� �radioactive and selected from a non‐ radioactive isotope of fluorine, iodine or bromine.  In a more preferred embodiment, the nuclide (Hal) is  selected from the group consisting of  ¹²⁷ I,  ¹⁹ F,  ⁷⁹ Br and  ⁸¹ Br.   In preferred embodiments, the PSMA targeting ligand a ccording to Formula (I), is one of the  following seven compounds:     

Formula (Ib)    Formula (Ic)     Formula (Id)   

Formula (Ie)  Formula (If)  Formula (Ig)   

Formula (Ih)    In other preferred embodiments, the PSMA targeting li gand according to Formula (I), is one of  the following compounds:   wherein iodine “I” is selected from  ¹²⁷ I,  ¹²⁵ I,  ¹²³ I,  ¹³¹ I, or  ¹²⁴ I.  In a most preferred embodiment, the PSMA targeting l igand according to Formula (I) is Il or Im.  The PSMA targeting ligands according to formula (I)  may be provided by suitable methods  known in the art.   In one aspect, the present invention relates to a m ethod for providing the PSMA targeting  ligands according to Formula (I) comprising the steps  of:  ^ Synthesis of a PSMA binding motif (BM)  ^ Coupling of linkers to BM    ● Coupling of BM‐linker to a chelator   ● Labeling the BM‐linker‐chelator with a halogen nuc lide, such as a halogen radionuclide   In one embodiment, the PSMA binding motif is Lys‐u rea‐Glu (LUG).  In one embodiment, the chelator is selected from:  1,4,7,10‐tetraazacyclododecane‐N,N',N',N"‐tetraacetic� �acid (DOTA),   N,N'‐bis(2‐hydroxy‐5‐(carboxyethyl)benzyl)ethylenedi amine N,N'‐diacetic acid (HBED‐CC),   14,7‐triazacyclononane‐1,4,7‐triacetic acid (NOTA),� �  2‐(4.7‐bis(carboxymethyl)‐1,4,7‐triazonan‐1‐yl)p entanedioic acid (NODAGA),   2‐(4,7,10‐tris(carboxymethyl)‐1,4,7,10‐tetraazacyclo dodecan‐1‐ yl)pentanedioic acid  (DOTAGA),   14,7‐triazacyclononane phosphinic acid (TRAP), 14,7‐ triazacyclononane‐1‐methyl(2‐ carboxyethyl)phosphinic acid‐4,7‐bis(methyl(2‐hydroxy methyl)phosphinic acid (NOPO),   3,6,9,15‐tetraazabicyclo9.3.1.pentadeca‐1 (15),11,13‐ triene‐3,6,9‐ triacetic acid (PCTA),   N'‐(5‐acetyl (hydroxy)aminopentyl‐N‐(5‐(4‐(5‐  aminopentyl)(hydroxy)amino‐4‐ oxobutanoyl)amino)pentyl‐N‐ hydroxysuccinamide (DFO),� �  diethylenetriaminepentaacetic acid (DTPA),   trans‐cyclohexyl‐diethylenetriaminepentaacetic acid (C HX‐DTPA),   1‐oxa‐4,7,10‐triazacyclododecane‐4,7,10‐triacetic� �acid (OXO‐Do3A),  p‐isothiocyanatobenzyl‐DTPA (SCN‐BZ‐DTPA),   1‐(p‐isothiocyanatobenzyl)‐3‐methyl‐DTPA (1B3M),� �  2‐(p‐isothiocyanatobenzyl)‐4‐methyl‐DTPA (1M3B),� �and  1‐(2)‐methyl‐4‐isocyanatobenzyl‐DTPA (MX‐DTPA).   In one embodiment, the halogen radionuclide is select ed from an isotope or radioisotope of  fluorine, iodine, bromine or astatine.  In a preferred embodiment, the halogen is a radionuc lide being one of the following  radioisotopes:  ¹⁸ F,  ¹²⁵ I,  ¹²³ I,  ¹³¹ I,  ¹²⁴ I,  ²¹¹ At,  ⁷⁷ Br and  ⁸⁰ Br.    In another preferred embodiment, the halogen is a no n‐radioactive nuclide selected from one  of the following isotopes:  ¹⁹ F,  ¹²⁷ I,  ⁷⁹ Br and  ⁸¹ Br.  The present invention also provides (Me) ₃ Sn precursors, silyl precursors, boron‐based  precursors, iodonium and diazonium salt precursors tha t can be used to provide the PSMA  targeting ligand according to Formula (I).   These precursors include precursors with and without  chelators and have the following  structures, here shown for the most preferred (Me) ₃ Sn precursors, but the same structures are  applicable for if substituting the (Me) ₃ Sn with silyl, boron, iodonium or diazonium:  Formula (II)     Formula (III)     

Formula (IV)  Formula (V)  Formula (VI)   

Formula (VII)  Formula (VIII)  and  Formula (IV)  A preferred method comprises the following steps:  ^ Synthesis of a PSMA binding motif (BM)  ^ Coupling of linkers to BM, wherein one or more of  the precursors of formula (II), (III),  (V) and (VII) is provided  ^ Coupling of BM‐linker to a chelator wherein one or  more of the precursors of Formula  (IV), (VI) and (VIII) is provided    ● Labeling the BM‐linker‐chelator with a halogen nuc lide, such as a halogen  radionuclide.  In a preferred embodiment, the PSMA binding motif is  Lys‐urea‐Glu (LUG).  The PSMA targeting ligands of formula (I) such as t he PSMA targeting ligands of formula (Ia),  (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii), (Ij) , (Ik), (Il), (Im) and (In) can be used in radioth erapy, as  imaging agents or as both i.e. as theranostic agents .  Surprisingly, despite the synthetic modifications in k ey regions, the binding profile and  biodistribution/pharmacokinetics of various of the here in disclosed new compounds are  comparable or superior to values observed for PSMA‐ 617 in direct head‐to‐head comparison.  This is particularly true for compounds Ii and Im.  PSMA‐617 is the most clinically applied  therapeutic PSMA inhibitor.   In one aspect, the PSMA targeting ligands of formula  (I) are for use in radiotherapy. In a  preferred embodiment the PSMA targeting ligands of fo rmula (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig),  (Ih), (Ii), (Ij), (Ik), (Il), (Im) and (In) are for  use in radiotherapy. In more preferred embodiments,� � compounds Ii or Im are used in radiotherapy. Prefera bly, when using ligands of formula (I) for  radiotherapy, the halogen isotope is selected from th e group consisting of  ²¹¹ At,  ¹²⁵ I,  ¹²³ I,  ⁷⁷ Br,  and  ⁸⁰ Br.  In another aspect, the PSMA targeting ligands of for mula (I) are for use in the treatment of  cancer, in particular prostate cancer. In a preferred  embodiment, the PSMA targeting ligands  of formula (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig),  (Ih), (Ii), (Ij), (Ik), (Il), (Im) and (In) are u sed in the  treatment of cancer, in particular prostate cancer. I n more preferred embodiments,  compounds Ii or Im are used in the treatment of ca ncer, in particular prostate cancer.  Preferably, when using ligands of formula (I) for ra diotherapy, the halogen isotope is  ²¹¹ At.  In yet another aspect, the PSMA targeting ligands of  formula (I) are for use as a theranostic  agent. In a preferred embodiment, the PSMA targeting� �ligands of formula (Ia), (Ib), (Ic), (Id),  (Ie), (If), (Ig), (Ih), (Ii), (Ij), (Ik), (Il), (Im)  and (In) are for use as theranostic agents. In mo re  preferred embodiments, compounds Ii or Im are for us e as a theranostic agents. Preferably,  when using ligands of formula (I) for theranostic ag ents, the halogen isotope is selected from  the group consisting of  ¹²⁵ I,  ¹²³ I,  ¹³¹ I,  ¹²⁴ I,  ⁷⁷ Br and  ⁸⁰ Br.  A further aspect of the invention is the use of PS MA targeting ligands of formula (I) as an  imaging agent.  In a preferred embodiment, the PSMA� �targeting ligands of formula (Ia), (Ib),  (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii), (Ij), (Ik) , (Il), (Im) and (In) are for use as imaging agent s. In more    preferred embodiments, compounds Ii or Im are for us e as imaging agents. Preferably, when  using ligands of formula (I) for theranostic agents,� �the halogen isotope is selected from the  group consisting of  ¹²⁵ I,  ¹²³ I,  ¹³¹ I,  ¹²⁴ I,  ⁷⁷ Br and  ⁸⁰ Br.  A further aspect of the invention is the use of PS MA targeting ligands of formula (I) as non‐ radioactive test‐compounds.  In a preferred embodime nt, the PSMA targeting ligands of  formula (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (I h), (Ii), (Ij), (Ik), (Il), (Im) and (In) are for  use as test‐ compounds. In more preferred embodiments, compounds Ii  or Im are for use test‐compound.  Preferably, when using ligands of formula (I) for th eranostic agents, the halogen isotope is  selected from the group consisting of  ¹²⁷ I,  ¹⁹ F,  ⁷⁹ Br or  ⁸¹ Br.    Examples  Example 1: GENERAL SYNTHETIC PROCDURES FOR THE SYNTHE SIS OF DOTA‐BEARING PSMA‐ TARGETING LIGANDS  Glu‐NCO ⁸ :  Firstly, di‐tert‐butyl L‐glutamate hydrochloride ( 6.76 mmol) was suspended in a 1:2 mixture of  dichloromethane (DCM) and saturated aqueous NaHCO ₃  (72 mL). The mixture was cooled to 0  °C and then triphosgene (3.38 mmol) was added. The� �reaction mixture was vigorously stirred  at 0 °C for 20 minutes, then warmed to room tempe rature, diluted with DCM and washed with  brine (2x30 mL). The organic layers were collected,  dried over Na ₂ SO ₄  and concentrated in  vacuo, affording the isocyanate Glu‐NCO.  PSMA binding motive ⁸ :  A solution of Glu‐NCO (6.73 mmol) in DCM (16 mL)� �was added to a mixture of tert‐butyl N ⁶ ‐ ((benzyloxy)carbonyl)‐L‐lysinate hydrochloride (7.40  mmol) and dry pyridine (7.40 mmol) in    DCM (50 mL). The reaction was stirred at room tempe rature 18 hours. Afterwards, the  reaction was diluted with 10 mL of DCM, washed with  0.1M HCl (5x15 mL) and washed with  brine (2x15 mL). The organic layers were collected,  dried over sodium sulphate and  evaporated in vacuo. Finally, the obtained crude was� �purified by flash chromatography.   The Cbz‐protected product (3.01 mmol) was mixed wit h Pd/C (10%) (0.31 mmol) catalyst,  dissolved in MeOH (15 mL) and hydrogen gas was bubb led through the mixture overnight. The  reaction mixture was filtered through a pad of diato maceous earth and the solvent was  evaporated in vacuo, yielding the PSMA binding motive .  PSMA binding motive‐linker:  The subsequent syntheses of the PSMA‐targeting pepti domimetics were carried out with the  following general procedure:  N‐Fmoc‐amino acid (0.21 mmol) and diisopropylethyla mine (DIPEA) (0.51 mmol) were  dissolved in dry dimethylformamide (DMF), 1‐[Bis(dime thylamino)methylene]‐1H‐1,2,3‐ triazolo[4,5‐b]pyridinium 3‐oxid hexafluorophosphate  (HATU) (0.31 mmol) was added to the  previous solution and stirred for 10 minutes. PSMA b inding motive (0.21 mmol) was dissolved  in dry DMF and dropwise added to the previous mixtu re for a total volume of 1 mL. The  reaction mixture was stirred at room temperature for� �4 to 24 hours depending on when  completion was attained.   Thereafter, the N‐terminus was deprotected by adding  piperidine (50% relative to DMF) and  stirring for 2 hours. Reaction mixture was poured ov er water and extracted with DCM. Organic  layers were dried over Na ₂ SO ₄  and volatiles removed in vacuo. The crude was  purified by flash  chromatography giving the desired free amine.   PSMA binding motive‐linker‐chelator:    To a PSMA binding motive‐linker construct solution  (0.2 mmol) in either DCM or DMF (1 mL),  trimethylamine (Et ₃ N) (0.31 mmol), DOTA‐mono‐NHS‐tris(tBu‐este r) (0.31 mmol) was added  and stirred for 18 hours at room temperature. The s ubsequent crude mixture was purified by  preparative HPLC.   PSMA binding motive‐linker‐chelator radiolabelling:  A tert‐butyl protected PSMA binding motive‐linker� �chelator construct was dissolved in a  solution of Chloramine‐T, methanol,  ²¹¹ At and acetic acid. The mixture was stirred an d reacted  during 30 minutes at room temperature. Afterwards, th e mixture was dried by use of a  nitrogen stream. The molecule was deprotected by addi tion of trifluoroacetic acid (TFA) and  heating to 60 °C for 30 minutes. Once fully deprot ected, the mixture was dried, redissolved in a  50:50 mixture of acetonitrile (MeCN)water and purified  by preparative HPLC.    Example 2: synthesis of PSMA‐targeting radiopharmaceu tical 1  Using the synthesis methods described in Example I,  the following PSMA‐targeting ligand was  provided:  Synthesis of PSMA binding motif following the procedu res previously described in Example I:   

  di-tert-butyl (S)-2-isocyanatopentanedioate   Di-tert-butyl L-glutamate hydrochloride (2.00 g, 6.76 mmol, 1.0 eq.) was suspended in DCM (24 mL) and sat. NaHCO3 (aq.) (48 mL). The mixture was cooled to 0 °C and then bis(trichloromethyl) carbonate (triphosgene) (1.00 g, 3.38 mmol, 0.5 eq.) was added (OBS: triphosgene is highly toxic and must be handled with extreme care). The reaction mixture was vigorously stirred at 0 °C for 20 minutes, then allowed to warm to room temperature, diluted with DCM (36 mL) and water (30 mL) and extracted with DCM (1 x 25 mL). The organic layers were washed with brine (1 x 20 mL), dried over Na2SO4 and concentrated in vacuo, affording the title compound as a transparent liquid (1.92 g, 6.73 mmol, quantitative). 1H-NMR (600 MHz, CDCl ₃ ) δ 3.97 (dd, J = 8.6, 4.5 Hz, 1H), 2.40 – 2.28 (m, 2H), 2.17 – 2.08 (m, 1H), 1.91 (m, 1H), 1.49 (d, J = 0.9 Hz, 9H), 1.44 (d, J = 0.9 Hz, 9H). ¹³ C NMR (151 MHz, CDCl3) δ 171.8, 170.1, 127.4, 83.7, 80.9, 57.5, 31.6, 29.2, 28.2. MS (ESI) m/z: 260.2 [M + 3H - CO] ⁺ (hydrolysed product) tri-tert-butyl (9S,13S)-3,11-dioxo-1-phenyl-2-oxa-4,10,12-triazapentadecane -9,13,15- tricarboxylate   A solution of di-tert-butyl (S)-2-isocyanatopentanedioate (1.92 g, 6.73 mmol, 1.0 eq.) in dry DCM (16 mL) was added to a mixture of tert-butyl N ⁶ -((benzyloxy)carbonyl)-L-lysinate hydrochloride (2.76 g, 7.40 mmol, 1.1 eq.) and dry pyridine (596 µL, 7.40 mmol, 1.1 eq.) in dry DCM (50 mL). The reaction was stirred at room temperature for 18 hours. Afterwards, the reaction was diluted with DCM (10 mL), washed with 0.1 M HCl(aq.) (5 x 15 mL) and brine (15 mL). The organic fraction was collected, dried over Na ₂ SO ₄ and evaporated in vacuo. The crude was purified by CombiFlash (heptane:EtOAc – 100:0 to 20:80) to isolate the title compound as a viscous colourless oil (2.93 g, 4.71 mmol, 70%). ¹ H NMR (400 MHz, CDCl3) δ 7.37- 7.28 (m, 5H), 5.22- 5.05 (m, 5H), 4.36-4.29 (m, 2H), 3.22- 3.12 (m, 2H), 2.35-2.22 (m, 2H), 2.09-2.02 (m, 1H), 1.87-1.67 (m, 3H), 1.66-1.23 (m, 31H) ¹³ C NMR (151 MHz, CDCl ₃ ) δ 172.6, 172.5, 171.3, 157.1, 156.8, 136.9, 128.6, 128.2, 82.2, 81.8, 80.6, 66.7, 53.4, 53.1, 40.8, 32.8, 32.0, 29.5, 29.1, 28.5, 28.2, 28.1, 22.8, 22.4. MS (ESI) m/z: 622.4 [M + H] ⁺ Rf (40 % EtOAc in heptane) = 0.42 di-tert-butyl (((S)-6-amino-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L-g lutamate To a flask containing tri-tert-butyl (9S,13S)-3,11-dioxo-1-phenyl-2-oxa-4,10,12- triazapentadecane-9,13,15-tricarboxylate (1.90 g, 3.06 mmol, 1.0 eq.) was added 10 wt. % Pd/C (325 mg, 0.31 mmol, 0.1 eq.) and suspended in MeOH (15 mL). The reaction vessel was purged with nitrogen and hydrogen gas was bubbled through the suspension overnight at atmospheric pressure (balloon). The reaction mixture was filtered over a pad of diatomaceous earth (Celite®) and volatiles removed in vacuo to yield di-tert-butyl (((S)-6-amino-1-(tert-butoxy)- 1-oxohexan-2-yl)carbamoyl)-L-glutamate as a viscous oil (1.49 g, 3.05 mmol, 99 %). ¹ H NMR (600 MHz, CDCl ₃ ) δ 5.23 (d, J = 8.0 Hz, 2H), 4.32 (q, J = 7.9, 4.9 Hz, 2H), 2.67 (t, J = 6.8 Hz, 2H), 2.37 – 2.22 (m, 2H), 2.10 – 2.01 (m, 1H), 1.87 – 1.72 (m, 1H), 1.65 – 1.57 (m, 1H),   1.55 – 1.24 (m, 31H). ¹³ C NMR (151 MHz, CDCl3) δ 172.7, 172.6, 172.4, 157.0, 82.1, 81.8, 80.7, 53.6, 53.2, 41.9, 33.2, 33.0, 31.8, 28.5, 28.20, 28.16, 28.13, 22.5. MS (ESI) m/z: 488.4 [M + H] ⁺ Coupling of linkers to the PSMA binding motive and  labelling with  ²¹¹ At following the  procedures previously described in Example I.  Example 3: Synthesis of PSMA‐targeting radiopharmaceu tical reference compounds  2,5‐dioxopyrrolidin‐1‐yl (1r,4r)‐4‐(((tert‐buto xycarbonyl)amino)methyl)cyclohexane‐1‐ carboxylate    Boc-tranexamic acid (3.00 g, 11.66 mmol) was dissolved in dry THF (100 mL). Thereafter EDC- HCl (3.36 g, 17.48 mmol) and N-hydroxysuccinimide (2.00 g, 17.48 mmol) were added as solids. The mixture was stirred at room temperature and the progress followed by UPLC-MS. The reaction mixture was a cloudy suspension that gradually became a clear solution over 4 hours of stirring. After 72 hours the reaction mixture was diluted with DCM (75 mL), washed with water (3x30 mL), dried over Mg ₂ SO ₄ , filtered and solvents evaporated under reduced pressure to yield 2,5‐dioxopyrrolidin‐1‐yl (1r,4r)‐4‐(((tert‐buto xycarbonyl)amino)methyl)cyclohexane‐1‐ carboxylate as a white powder (2.6 g, 7.25 mmol, 63%). ¹ H NMR (400 MHz, CDCl3) δ 2.99 (t, J = 6.5 Hz, 2H), 2.82 (s, 4H), 2.58 (tt, J = 12.2, 3.6 Hz, 1H), 2.17 (dd, J = 13.9, 3.6 Hz, 2H), 1.88 (dd, J = 13.7, 3.5 Hz, 2H), 1.60 (dd, J = 13.0, 3.4 Hz, 2H), 1.55 (d, J = 2.8 Hz, 1H), 1.44 (s, 9H), 1.01 (qd, J = 13.2, 3.6 Hz, 2H). ¹³ C NMR (101 MHz, CDCl3) δ 170.9, 169.3, 156.2, 79.4, 46.5, 40.8, 37.6, 29.5, 28.5, 28.4, 25.7. MS (ESI) m/z: 255.3 [M + H - Boc] ⁺ General procedure I: Fmoc solution-phase synthesis of LuG-linker molecules N-Fmoc-amino acid (Fmoc-AA) (1.0 eq) and DIPEA (2.5 eq) were dissolved in dry DMF (1-3 mL), HATU (1.5 eq) was added to the previous solution and stirred for 15 minutes. The corresponding amine (1.0 eq) was dissolved in dry DMF (1-3 mL) and added to the previous mixture for a total volume of 2-6 mL. The reaction mixture was stirred at room temperature, until completion (5 to 24 hours). Thereafter, the Fmoc-protected N-terminus was deprotected by adding piperidine (50% vol. relative to DMF) and stirred for an additional 2 hours. Then, the reaction mixture was poured over water (10 mL) and extracted with DCM (2 x 15 mL). The   combined organic layers were washed with water (3 x 10 mL), dried over MgSO ₄ and volatiles removed in vacuo. The crude was purified by CombiFlash giving the desired free amine. General procedure II: synthesis of tranexamic acid-peptide conjugates Zwitter ionic alpha-amino acid (1.2 eq.) and Na ₂ CO ₃ (2.0 eq.) were dissolved in 20 mL of a 1:2 mixture of H ₂ O/1,4-dioxane. Afterwards, a solution of 2,5‐dioxopyrrolidin‐1‐yl (1r,4r)‐4‐(((tert‐ butoxycarbonyl)amino)methyl)cyclohexane‐1‐carboxylate (1.0 eq.) in 1,4-dioxane (5 mL) was added. The mixture was stirred for 4 hours, where completion was observed by UPLC-MS. Thereafter, the pH of the mixture was adjusted to 3 with 1M HCl _(aq.) and the precipitated product was extracted with EtOAc (3x15mL), dried over Mg ₂ SO ₄ , filtered and solvents evaporated in vacuo to yield the desired compound as a white powder. General procedure III: synthesis of Boc-LuG-linker molecules Carboxylic acid (1.0 eq) and DIPEA (2.5 eq) were dissolved in dry DMF (1-3 mL), HATU (1.5 eq) was added to the previous solution and stirred for 15 minutes.12 (1.0 eq) was dissolved in dry DMF (1-3 mL) and added to the previous mixture for a total volume of 2-6 mL. The reaction mixture was stirred at room temperature for 5 to 24 hours depending on when completion was attained (followed by UPLC-MS). Once completed, the mixture was poured into 20 mL of water and extracted with DCM (3x15 mL). The organic fractions were washed once more with water (50 mL) to fully remove the DMF, dried over magnesium sulfate, filtered and solvents removed in vacuo. Crude was purified by CombiFlash (Heptane:EtOAc – 100:0 to 0:100 with a gradual increase of 20% EtOAc every 6 minutes). Fractions containing desired product were combined and volatiles removed under reduced pressure to obtain the compounds. General procedure IV: deprotection of Boc-LUG-linker molecules ^t Bu-Boc-protected molecule was dissolved in a 1:1 mixture of TFA:DCM (5 mL). The solution was stirred at room temperature for 2 hours while being monitored by UPLC-MS. Once the deprotection was complete the mixture was evaporated under reduced pressure. Thereafter, the compounds were left under high vacuum for 72 hours to fully remove TFA traces. General procedures V and VI: synthesis of DOTA-linker-LUG compounds OBS: materials used to handle DOTA molecules are all free from metal, either glass or plastic, to avoid potential undesired chelation. V – for Fmoc route: To an LuG-linker solution (1 eq.) in DCM, Et3N (6 eq.), DOTA-mono-NHS- tris( ^t Bu-ester) (1.2 eq.) were added and stirred overnight (~16 hours). Afterwards the reaction mixture was evaporated under reduced pressure and the resulting crude re-dissolved in 1:1 mixture of TFA/DCM (3 mL) and mechanically shaken for 3 hours. Thereafter, volatiles were removed in vacuo and the oily crude purified by preparatory HPLC. Fractions containing desired product were collected and lyophilized to obtain the compounds as white solids.   VI – for Boc route: To a LuG-linker solution (1 eq.) in DMF, Et ₃ N (6 eq.), DOTA-mono-NHS- ester (1.2 eq.) were added and stirred overnight (~16 hours). Afterwards the reaction mixture was placed under a stream of air until dryness. Thereafter, the oily crude was purified by preparatory HPLC. Fractions containing desired product were collected and lyophilized to obtain the compounds as white solids. di-tert-butyl (((S)-6-((S)-2-amino-3-(naphthalen-2-yl)propanamido)-1-(tert -butoxy)-1- oxohexan-2-yl)carbamoyl)-L-glutamate di-tert-butyl (((S)-6-((S)-2-amino-3-(naphthalen-2-yl)propanamido)-1-(tert -butoxy)-1-oxohexan-2- yl)carbamoyl)-L-glutamate was prepared from di-tert-butyl (((S)-6-amino-1-(tert-butoxy)-1- oxohexan-2-yl)carbamoyl)-L-glutamate (500 mg, 1.02 mmol) following general procedure I employing (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(naphtha len-2-yl)propanoic acid (Fmoc-3-(2-naphthyl)-L-alanine) (449 mg, 1.02 mmol) as the Fmoc-AA and reacting for 15 hours. The crude was purified by CombiFlash, impurities were washed off the column with 100% EtOAc and desired compound was eluted (DCM:MeOH – 100:0 to 90:10). Fractions containing desired product were combined and volatiles removed under reduced pressure to obtain the compound as a white semisolid (352 mg, 0.51 mmol, 50%). ¹ H NMR (600 MHz, CDCl ₃ ) δ 7.98 (s, 1H), 7.80 – 7.73 (m, 3H), 7.67 (s, 1H), 7.52 (t, J = 5.9 Hz, 1H), 7.46 – 7.41 (m, 2H), 7.34 (dd, J = 8.4, 1.7 Hz, 1H), 5.95 (s, 1H), 5.88 (d, J = 8.1 Hz, 1H), 4.30 – 4.26 (m, 1H), 4.24 – 4.16 (m, 1H), 4.14 – 4.07 (m, 1H), 3.36 – 3.30 (m, 1H), 3.30 – 3.23 (m, 1H), 3.13 (dd, J = 13.8, 7.8 Hz, 1H), 3.04 – 2.97 (m, 1H), 2.35 – 2.25 (m, 2H), 2.08 – 2.01 (m, 2H), 1.87 – 1.77 (m, 2H), 1.69 – 1.61 (m, 1H), 1.57 – 1.51 (m, 1H), 1.41 (s, 9H), 1.40 (d, J = 1.6 Hz, 18H), 1.37 – 1.31 (m, 1H), 0.92 – 0.75 (m, 2H). ¹³ C NMR (151 MHz, CDCl3) δ 173.2, 172.9, 172.6, 157.8, 133.5, 133.3, 132.6, 128.6, 128.4, 127.7, 127.7, 127.3, 126.3, 125.9, 82.1, 81.5, 80.7, 53.3, 53.1, 39.4, 38.9, 31.8, 31.7, 31.6, 28.5, 28.3, 28.1, 28.0, 22.1. MS (ESI) m/z: 685.5 [M + H] ⁺ Rf (10 % MeOH in DCM, with tailing) = 0.34 di-tert-butyl (((S)-6-((S)-2-amino-3-(2-iodophenyl)propanamido)-1-(tert-bu toxy)-1- oxohexan-2-yl)carbamoyl)-L-glutamate  

di-tert-butyl (((S)-6-((S)-2-amino-3-(2-iodophenyl)propanamido)-1-(tert-bu toxy)-1-oxohexan-2- yl)carbamoyl)-L-glutamate was prepared from di-tert-butyl (((S)-6-amino-1-(tert-butoxy)-1- oxohexan-2-yl)carbamoyl)-L-glutamate (250 mg, 0.51 mmol) following general procedure I employing (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2-iodop henyl)propanoic acid (Fmoc-2-iodo-L-phenylalanine) (263 mg, 0.51 mmol) as the Fmoc-AA and reacting for 5 hours. The crude was purified by CombiFlash, impurities were washed off the column with 100% EtOAc and desired compound was eluted (DCM:MeOH – 100:0 to 90:10). Fractions containing desired product were combined and volatiles removed under reduced pressure to obtain the compound as a yellowish oil (80 mg, 0.10 mmol, 21%). ¹ H NMR (400 MHz, CDCl3) δ 7.83 (d, J = 7.9 Hz, 1H), 7.32 – 7.15 (m, 2H), 6.92 (td, J = 7.4, 2.1 Hz, 1H), 5.46 – 5.34 (m, 2H), 4.31 (m, 3H), 3.67 (m, 1H), 3.49 – 3.36 (m, 1H), 3.25 (m, 2H), 2.86 (dd, J = 14.0, 7.1 Hz, 1H), 2.40 – 2.20 (m, 2H), 2.11 – 2.00 (m, 1H), 1.85 – 1.70 (m, 3H), 1.74 – 1.57 (m, 4H), 1.56 – 1.30 (m, 31H). ¹³ C NMR (101 MHz, CDCl3) δ 174.1, 172.58, 172.56, 172.4, 157.2, 141.2, 139.9, 130.83, 130.79, 128.68, 128.63, 128.60, 101.4, 82.0, 81.7, 80.6, 55.9, 53.5, 53.1, 45.6, 38.7, 32.4, 31.8, 29.1, 28.6, 28.2,22.4. MS (ESI) m/z: 761.3 [M + H] ⁺ R _f (10 % MeOH in DCM, with tailing) = 0.39 di-tert-butyl (((S)-6-((S)-2-((1r,4S)-4-(aminomethyl)cyclohexane-1-carboxa mido)-3- (naphthalen-2-yl)propanamido)-1-(tert-butoxy)-1-oxohexan-2-y l)carbamoyl)-L-glutamate di-tert-butyl (((S)-6-((S)-2-((1r,4S)-4-(aminomethyl)cyclohexane-1-carboxa mido)-3-(naphthalen- 2-yl)propanamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl) -L-glutamate was prepared from   di-tert-butyl (((S)-6-((S)-2-amino-3-(naphthalen-2-yl)propanamido)-1-(tert -butoxy)-1-oxohexan-2- yl)carbamoyl)-L-glutamate (180 mg, 0.26 mmol) following general procedure I employing (1r,4r)-4-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl )cyclohexane-1-carboxylic acid (Fmoc-tranexamic acid) (100 mg, 0.26 mmol) as the Fmoc-AA and reacting for 15 hours. The crude was purified by CombiFlash, impurities were washed off the column with 100% EtOAc and desired compound was eluted (DCM:MeOH – 100:0 to 90:10). Fractions containing desired product were combined and volatiles removed under reduced pressure to obtain the compound as a yellowish oil (85 mg, 0.10 mmol, 40%). ¹ H NMR (400 MHz, CDCl3) δ 8.45 (s, 1H), 7.83 (d, J = 8.3 Hz, 1H), 7.67 – 7.61 (m, 2H), 7.54 (d, J = 8.1 Hz, 1H), 7.42 (t, J = 7.5 Hz, 1H), 7.34 (t, J = 7.4 Hz, 1H), 7.18 – 7.07 (m, 2H), 7.01 (d, 1H), 6.17 (d, J = 8.7 Hz, 1H), 5.21 – 5.16 (m, 1H), 4.68 – 4.57 (m, 1H), 4.32 – 4.22 (m, 1H), 3.55 – 3.41 (m, 2H), 3.20 (dd, J = 13.8, 4.1 Hz, 1H), 3.13 – 2.98 (m, 2H), 2.49 – 2.30 (m, 3H), 2.24 – 2.10 (m, 1H), 1.97 – 1.79 (m, 2H), 1.79 – 1.68 (m, 2H), 1.57 (s, 8H), 1.46 – 1.35 (m, 27H), 1.20 – 1.06 (m, 3H), 0.99 – 0.60 (m, 4H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ 177.26, 174.82, 174.20, 172.52, 157.56, 135.06, 133.35, 132.20, 128.05, 127.85, 127.30, 125.91, 125.36, 82.46, 80.93, 80.39, 54.60, 53.26, 52.28, 50.34, 48.12, 44.63, 40.42, 39.67, 39.45, 32.77, 31.65, 29.98, 29.08, 28.90, 28.21, 28.16, 28.07, 26.90, 25.68, 24.71, 23.42. MS (ESI) m/z: 825.7 [M + H] ⁺ R _f (10 % MeOH in DCM, with tailing) = 0.28 di-tert-butyl (((S)-6-((S)-2-((1r,4S)-4-(aminomethyl)cyclohexane-1-carboxa mido)-3-(2- iodophenyl)propanamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carb amoyl)-L-glutamate di-tert-butyl (((S)-6-((S)-2-((1r,4S)-4-(aminomethyl)cyclohexane-1-carboxa mido)-3-(2- iodophenyl)propanamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carb amoyl)-L-glutamate was prepared from di-tert-butyl (((S)-6-((S)-2-amino-3-(2-iodophenyl)propanamido)-1-(tert-bu toxy)-1- oxohexan-2-yl)carbamoyl)-L-glutamate (75 mg, 0.10 mmol) following general procedure I employing (1r,4r)-4-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl )cyclohexane-1- carboxylic acid (Fmoc-tranexamic acid) (38 mg, 0.10 mmol) as the Fmoc-AA and reacting for 7 hours. The crude was purified by CombiFlash, impurities were washed off the column with 100% EtOAc and desired compound was eluted (DCM:MeOH – 100:0 to 90:10). Fractions   containing desired product were combined and volatiles removed under reduced pressure to obtain the compound as a white semisolid (70 mg, 0.08 mmol, 76%). R _f (10 % MeOH in DCM, with tailing) = 0.25 ¹ H NMR (400 MHz, CDCl3) δ 7.74 (dd, J = 7.8, 2.7 Hz, 1H), 7.30 (d, J = 7.2 Hz, 1H), 7.19 (t, J = 7.6 Hz, 1H), 6.87 (td, J = 7.6, 1.7 Hz, 1H), 4.87 (q, J = 7.8 Hz, 1H), 4.46 – 4.31 (m, 1H), 4.24 – 4.13 (m, 1H), 3.44 – 3.26 (m, 1H), 3.24 – 3.10 (m, 2H), 3.05 (q, J = 7.3 Hz, 3H), 3.00 – 2.89 (m, 1H), 2.88 – 2.74 (m, 2H), 2.43 – 2.24 (m, 2H), 2.21 – 2.02 (m, 2H), 1.97 – 1.81 (m, 3H), 1.81 – 1.60 (m, 5H), 1.60 – 1.48 (m, 2H), 1.48 – 1.39 (m, 27H), 1.28 – 1.10 (m, 1H), 1.07 – 0.91 (m, 2H). MS (ESI) m/z: 900.2 [M + H] ⁺ Rf (10 % MeOH in DCM, with tailing) = 0.25 di-tert-butyl (((S)-6-((S)-2-((S)-2-amino-3-(4-iodophenyl)propanamido)-3-( naphthalen-2- yl)propanamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L -glutamate di-tert-butyl (((S)-6-((S)-2-((S)-2-amino-3-(4-iodophenyl)propanamido)-3-( naphthalen-2- yl)propanamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L -glutamate was prepared from di- tert-butyl (((S)-6-((S)-2-amino-3-(naphthalen-2-yl)propanamido)-1-(tert -butoxy)-1-oxohexan-2- yl)carbamoyl)-L-glutamate (40 mg, 0.58 mmol) following general procedure I employing (S)-2- ((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-iodophenyl) propanoic acid (Fmoc-4-iodo-L- phenylalanine) (30 mg, 0.58 mmol) as the Fmoc-AA and reacting for 17 hours. The crude was purified by CombiFlash, impurities were washed off the column with 100% EtOAc and desired compound was eluted (DCM:MeOH – 100:0 to 90:10). Fractions containing desired product were combined and volatiles removed under reduced pressure to obtain the compound as a white semisolid (20 mg, 0.02 mmol, 37%). ¹ H NMR (600 MHz, CDCl ₃ ) δ 7.79 – 7.68 (m, 4H), 7.65 (d, J = 6.9 Hz, 1H), 7.43 (dd, J = 6.3, 3.4 Hz, 3H), 7.37 (d, J = 7.8 Hz, 2H), 7.34 (s, 1H), 6.68 (d, J = 8.0, 4.3 Hz, 1H), 6.01 (s, 1H), 4.81 (s, 1H), 4.40 – 4.29 (m, 1H), 4.27 – 4.18 (m, 1H), 3.89 – 3.66 (m, 1H), 3.35 (dd, J = 13.7, 6.0 Hz, 2H), 3.27 (q, J = 7.3, 5.5 Hz, 1H), 3.23 – 3.14 (m, 1H), 3.05 – 2.95 (m, 1H), 2.90 (dd, J = 13.9, 5.6 Hz, 1H), 2.70 – 2.50 (m, 1H), 2.37 – 2.29 (m, 2H), 2.12 – 2.01 (m, 1H), 1.90 – 1.76 (m, 2H), 1.62 (dt, J = 33.8, 9.4 Hz, 3H), 1.46 – 1.34 (m, 27H), 1.31 – 1.18 (m, 4H). MS (ESI) m/z: 958.4 [M + H] ⁺ R _f (10 % MeOH in DCM, with tailing) = 0.27   di-tert-butyl (((S)-6-((S)-2-((1S,4S)-4-(((S)-2-amino-3-(4- iodophenyl)propanamido)methyl)cyclohexane-1-carboxamido)-3-( naphthalen-2- yl)propanamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L -glutamate di-tert-butyl (((S)-6-((S)-2-((1S,4S)-4-(((S)-2-amino-3-(4- iodophenyl)propanamido)methyl)cyclohexane-1-carboxamido)-3-( naphthalen-2- yl)propanamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L -glutamate was prepared from di- tert-butyl (((S)-6-((S)-2-((1r,4S)-4-(aminomethyl)cyclohexane-1-carboxa mido)-3-(naphthalen-2- yl)propanamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L -glutamate (100 mg, 0.12 mmol) following general procedure I employing (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3- (4-iodophenyl)propanoic acid (Fmoc-4-iodo-L-alanine) (62 mg, 0.12 mmol) as the Fmoc-AA and reacting for 16 hours. Compound was purified by CombiFlash (Heptane:EtOAc:DCM:MeOH – 100:0:0:0 to 0:100:0:0 and then 0:0:100:0 to 0:0:90:10). Fractions containing desired product were combined and volatiles removed under reduced pressure to obtain the compound as a white semisolid (75 mg, 0.07 mmol, 56%). ¹ H NMR (400 MHz, CDCl3) δ 7.72 – 7.53 (m, 5H), 7.44 (t, 1H), 7.39 (t, 1H), 7.21 (t, J = 6.1 Hz, 1H), 7.13 (d, J = 8.3 Hz, 1H), 6.95 (dd, J = 11.7, 8.1 Hz, 2H), 6.12 (d, J = 8.5 Hz, 1H), 5.18 (s, 1H), 4.64 (s, 1H), 4.30 (dd, J = 9.7, 5.4 Hz, 1H), 3.62 – 3.38 (m, 4H), 3.22 (dd, J = 13.7, 4.3 Hz, 1H), 3.15 (dd, J = 13.8, 4.2 Hz, 3H), 3.10 – 2.99 (m, 4H), 2.98 – 2.88 (m, 1H), 2.64 (dd, J = 13.8, 8.9 Hz, 2H), 2.50 – 2.32 (m, 4H), 2.27 – 2.12 (m, 1H), 1.91 (tdd, J = 14.8, 8.1, 3.5 Hz, 1H), 1.69 (d, J = 40.8 Hz, 2H), 1.59 (s, 8H), 1.46 – 1.37 (m, 27H), 1.30 – 1.17 (m, 2H), 1.17 – 0.95 (m, 1H), 0.93 – 0.63 (m, 1H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ 177.22, 174.96, 173.77, 172.54 , 157.58, 137.81, 137.66, 135.08, 133.42, 132.27, 131.50, 131.44, 128.16, 127.96, 127.88, 127.38, 127.07, 126.03, 125.47, 92.19, 82.60, 81.02, 80.48, 56.38, 54.67, 53.34, 52.33, 45.16, 44.52, 40.51, 37.51, 37.07, 31.97, 30.19, 29.65, 29.11, 28.90, 28.29, 28.20, 28.14, 27.96, 26.98, 24.78, 22.78. MS (ESI) m/z: 1098.3 [M + H] ⁺ R _f (10 % MeOH in DCM, with tailing) = 0.21 (S)-2-((1r,4S)-4-(((tert-butoxycarbonyl)amino)methyl)cyclohe xane-1-carboxamido)-3-(4- iodophenyl)propanoic acid   (S)-2-((1r,4S)-4-(((tert-butoxycarbonyl)amino)methyl)cyclohe xane-1-carboxamido)-3-(4- iodophenyl)propanoic acid was prepared following general procedure II, employing (S)-2- amino-3-(4-iodophenyl)propanoic acid (4-iodo-L-phenylalanine) as the alpha-amino acid (390 mg, 0.74 mmol, 86%) ¹ H NMR (600 MHz, CD3OD) δ 7.61 (dt, J = 8.3, 1.9 Hz, 2H), 7.01 (dt, J = 8.3, 1.8 Hz, 2H), 4.64 (q, J = 9.2 Hz, 1H), 3.17 (dd, J = 14.0, 5.0 Hz, 1H), 2.93 – 2.85 (m, 4H), 2.12 (tt, J = 12.2, 3.5 Hz, 1H), 1.84 – 1.74 (m, 5H), 1.67 – 1.60 (m, 1H), 1.43 (s, 9H), 0.99 – 0.88 (m, 2H). MS (ESI) m/z: 529.2 [M - H]- (S)-2-((1r,4S)-4-(((tert-butoxycarbonyl)amino)methyl)cyclohe xane-1-carboxamido)-3-(3- iodophenyl)propanoic acid (S)-2-((1r,4S)-4-(((tert-butoxycarbonyl)amino)methyl)cyclohe xane-1-carboxamido)-3-(3- iodophenyl)propanoic acid was prepared following general procedure II, employing (S)-2- amino-3-(3-iodophenyl)propanoic acid (3-iodo-L-phenylalanine) as the alpha-amino acid (390 mg, 0.74 mmol, 86%) ¹ H NMR (400 MHz, CD ₃ OD) 7.61 (dt, J = 8.3, 1.9 Hz, 2H), 7.01 (dt, J = 8.3, 1.8 Hz, 2H), 4.64 (q, J = 9.2 Hz, 1H), 3.17 (dd, J = 14.0, 5.0 Hz, 1H), 2.93 – 2.85 (m, 4H), 2.12 (tt, J = 12.2, 3.5 Hz, 1H), 1.84 – 1.74 (m, 5H), 1.67 – 1.60 (m, 1H), 1.43 (s, 9H), 0.99 – 0.88 (m, 2H). ¹³ C NMR (101 MHz, MeOD) δ 178.7, 174.4, 158.6, 141.2, 139.5, 136.9, 131.2, 129.7, 94.8, 79.8, 54.3, 46.0, 39.0, 37.8, 30.8, 29.8, 28.8, 26.3. MS (ESI) m/z: 529.3 [M - H]- (R)-2-((1r,4R)-4-(((tert-butoxycarbonyl)amino)methyl)cyclohe xane-1-carboxamido)-3-(5- iodo-1H-indol-3-yl)propanoic acid   (R)-2-((1r,4R)-4-(((tert-butoxycarbonyl)amino)methyl)cyclohe xane-1-carboxamido)-3-(5-iodo- 1H-indol-3-yl)propanoic acid was prepared following general procedure II, employing 2-amino- 3-(5-iodo-1H-indol-3-yl)propanoic acid as the alpha-amino acid, the compound was purified by CombiFlash (Heptane:EtOAc:AcOH – 100:0:0.5 to 15:85:0.5) (100 mg, 0.17 mmol, 46%) Rf (40 % EtOAc in heptane, with tailing) = 0.21 ¹ H NMR (400 MHz, CD ₃ OD) δ 10.53 (s, 1H), 7.90 (d, J = 1.6 Hz, 1H), 7.34 (dd, J = 8.5, 1.6 Hz, 1H), 7.16 (d, J = 8.5 Hz, 1H), 7.08 (d, J = 2.0 Hz, 1H), 4.70 (dd, J = 7.8, 4.8 Hz, 1H), 4.11 (q, J = 7.1 Hz, 1H), 3.35 – 3.26 (m, 2H), 3.14 (dd, J = 14.7, 7.9 Hz, 1H), 2.87 (d, J = 6.7 Hz, 2H), 2.13 (tt, J = 12.2, 3.5 Hz, 1H), 1.86 – 1.73 (m, 3H), 1.74 – 1.65 (m, 1H), 1.44 (s, 9H), 1.02 – 0.86 (m, 2H). ¹³ C NMR (101 MHz, CD ₃ OD) δ 178.7, 175.0, 158.7, 137.1, 131.8, 130.7, 128.4, 125.7, 114.5, 110.9, 82.8, 79.8, 54.6, 46.0, 39.1, 30.9, 30.0, 28.8, 28.2. MS (ESI) m/z: 568.2 [M - H]- di-tert-butyl (((S)-1-(tert-butoxy)-6-((S)-2-((1r,4S)-4-(((tert- butoxycarbonyl)amino)methyl)cyclohexane-1-carboxamido)-3-(4- iodophenyl)propanamido)-1-oxohexan-2-yl)carbamoyl)-L-glutama te di-tert-butyl (((S)-1-(tert-butoxy)-6-((S)-2-((1r,4S)-4-(((tert- butoxycarbonyl)amino)methyl)cyclohexane-1-carboxamido)-3-(4- iodophenyl)propanamido)-1- oxohexan-2-yl)carbamoyl)-L-glutamate was prepared following general procedure III, employing (S)-2-((1r,4S)-4-(((tert-butoxycarbonyl)amino)methyl)cyclohe xane-1-carboxamido)-3- (4-iodophenyl)propanoic acid as the carboxylic acid and reacting for 5 hours. Compound was obtained as a white/yellow solid (296 mg, 0.29 mmol, 63%). ¹ H NMR (400 MHz, CDCl ₃ , mixture of un-assigned rotamers) δ 7.56 (d, J = 8.0 Hz, 2H), 7.48 (d, J = 7.9 Hz, 2H), 6.96 – 6.86 (m,   2H), 6.67 – 6.60 (m, 1H), 6.01 – 5.97 (m, 1H), 5.63 – 5.57 (m, 1H), 5.48 – 5.40 (m, 1H), 4.97 (s, 1H), 4.70 – 4.50 (m, 1H), 4.39 – 4.17 (m, 2H), 3.28 – 3.14 (m, 1H), 3.09 – 2.95 (m, 1H), 2.98 – 2.86 (m, 4H), 2.89 – 2.77 (m, 1H), 2.43 – 2.21 (m, 2H), 2.16 – 1.98 (m, 1H), 2.05 – 2.01 (m, 1H), 1.98 –431.94 (m, 2H), 1.92 – 1.68 (m, 1H), 1.81 – 1.77 (m, 7H), 1.47 – 1.40 (m, 36H), 1.38 – 1.29 (m, 1H), 1.28 – 1.20 (m, 1H), 0.98 – 0.75 (m, 2H). ¹³ C NMR (101 MHz, CDCl3, mixture of un-assigned rotamers) δ 176.23, 172.92, 172.70, 172.66, 172.56, 172.50, 171.54, 162.70, 157.66, 157.54, 157.32, 157.28, 156.23, 137.61, 137.44, 136.69, 131.55, 131.44, 92.27, 92.04, 82.57, 82.29, 82.21, 81.82, 81.69, 81.61, 81.09, 80.75, 80.55, 79.18, 77.36, 60.51, 54.35, 54.29, 53.48, 53.26, 53.17, 53.04, 52.43, 46.71, 45.48, 45.14, 44.65, 38.63, 38.18, 36.61, 32.58, 32.00, 31.78, 31.75, 29.98, 29.85, 29.77, 29.44, 29.20, 29.12, 28.86, 28.74, 28.56, 28.45, 28.24, 28.21, 28.15, 22.55, 22.14 MS (ESI) m/z: 1001.5 [M + H] ⁺ R _f (60 % EtOAc in heptane) = 0.32 di-tert-butyl (((S)-1-(tert-butoxy)-6-((S)-2-((1r,4S)-4-(((tert- butoxycarbonyl)amino)methyl)cyclohexane-1-carboxamido)-3-(3- iodophenyl)propanamido)-1-oxohexan-2-yl)carbamoyl)-L-glutama te di-tert-butyl (((S)-1-(tert-butoxy)-6-((S)-2-((1r,4S)-4-(((tert- butoxycarbonyl)amino)methyl)cyclohexane-1-carboxamido)-3-(3- iodophenyl)propanamido)-1- oxohexan-2-yl)carbamoyl)-L-glutamate was prepared following general procedure III, employing (S)-2-((1r,4S)-4-(((tert-butoxycarbonyl)amino)methyl)cyclohe xane-1-carboxamido)-3- (3-iodophenyl)propanoic acid as the carboxylic acid and reacting for 24 hours. Compound was obtained as an off-white solid (210 mg, 0.21 mmol, 56%). ¹ H NMR (400 MHz, CDCl3, mixture of un-assigned rotamers) δ 7.59 – 7.47 (m, 2H), 7.05 – 6.94 (m, 1H), 6.70 – 6.62 (m, 1H), 6.03 – 5.97 (m, 1H), 4.94 – 4.90 (m, 1H), 4.67 – 4.55 (m, 2H), 4.35 – 4.23 (m, 1H), 3.51 – 3.43 (m, 1H), 3.03 – 2.85 (m, 4H), 2.81 – 2.77 (m, 7H), 2.45 – 2.26 (m, 2H), 2.22 – 1.98 (m, 1H), 1.92 – 1.70 (m, 2H), 1.69 – 1.61 (m, 1H), 1.58 – 1.54 (m, 6H), 1.50 – 1.39 (m, 38H), 1.36 – 1.20 (m, 1H), 0.98 – 0.84 (m, 1H), 0.84 – 0.77 (m, 1H). ¹³ C NMR (101 MHz, CDCl ₃ , mixture of un- assigned rotamers) δ 177.36, 176.09, 175.08, 172.63, 172.58, 172.54, 172.49, 157.56, 157.26, 157.24, 156.15, 140.08, 139.54, 138.47, 138.28, 135.86, 130.32, 130.18, 128.73, 128.41, 94.17, 82.80, 82.22, 82.12, 81.78, 81.63, 81.04, 80.67, 80.48, 79.16, 77.36, 54.73,   53.42, 53.26, 53.16, 52.35, 46.74, 45.51, 44.66, 38.88, 38.73, 37.57, 31.75, 31.73, 30.07, 29.99, 29.88, 29.69, 29.59, 29.21, 29.07, 28.86, 28.55, 28.47, 28.41, 28.24, 28.21, 28.17, 28.15. MS (ESI) m/z: 1001.5 [M + H] ⁺ R _f (60 % EtOAc in heptane) = 0.38 di-tert-butyl (((S)-1-(tert-butoxy)-6-((R)-2-((1r,4R)-4-(((tert- butoxycarbonyl)amino)methyl)cyclohexane-1-carboxamido)-3-(5- iodo-1H-indol-3- yl)propanamido)-1-oxohexan-2-yl)carbamoyl)-L-glutamate di-tert-butyl (((S)-1-(tert-butoxy)-6-((R)-2-((1r,4R)-4-(((tert- butoxycarbonyl)amino)methyl)cyclohexane-1-carboxamido)-3-(5- iodo-1H-indol-3- yl)propanamido)-1-oxohexan-2-yl)carbamoyl)-L-glutamate was prepared following general procedure III, employing (R)-2-((1r,4R)-4-(((tert-butoxycarbonyl)amino)methyl)cyclohe xane-1- carboxamido)-3-(5-iodo-1H-indol-3-yl)propanoic acid as the carboxylic acid and reacting for 10 hours. Compound was obtained as a white semisolid (80 mg, 0.08 mmol, 45%). ¹ H NMR (400 MHz, CDCl ₃ , mixture of un-assigned rotamers) δ 9.70 (s, 1H), 8.99 (s, 1H), 8.01 (d, J = 3.7 Hz, 1H), 7.91 (d, J = 1.5 Hz, 1H), 7.44 – 7.34 (m, 1H), 7.22 (d, J = 8.5 Hz, 1H), 7.13 (d, J = 8.5 Hz, 1H), 7.02 – 6.95 (m, 1H), 6.60 (d, J = 7.6 Hz, 1H), 6.20 (s, 1H), 5.80 (s, 1H), 5.70 (d, J = 8.3 Hz, 1H), 5.42 – 5.31 (m, 1H), 4.34 – 4.29 (m, 1H), 4.20 – 4.12 (m, 1H), 3.16 – 3.08 (m, 1H), 3.03 – 2.91 (m, 3H), 2.40 – 2.27 (m, 2H), 2.10 – 2.01 (m, 1H), 1.92 – 1.83 (m, 1H), 1.83 – 1.80 (m, 2H), 1.79 – 1.71 (m, 2H), 1.49 – 1.40 (m, 45H), 1.30 – 1.21 (m, 3H), 1.21 – 1.11 (m, 1H), 0.97 – 0.83 (m, 4H). ¹³ C NMR (101 MHz, CDCl3, mixture of un-assigned rotamers) δ 176.58, 175.89, 173.80, 172.68, 172.65, 172.59, 171.62, 157.76, 157.63, 156.26, 135.60, 135.41, 130.34, 130.16, 130.06, 127.67, 127.56, 124.80, 124.36, 113.82, 113.56, 110.12, 109.74, 82.97, 82.85, 82.36, 82.33, 81.95, 81.44, 80.90, 80.71, 79.23, 77.36, 60.52, 54.12, 53.90, 53.58, 53.45, 53.31, 52.86, 46.74, 45.22, 44.75, 39.05, 38.76, 37.79, 32.32, 31.87, 31.84, 29.93, 29.75, 29.21, 28.69, 28.57, 28.45, 28.22, 28.16, 28.14, 22.55, 21.16. MS (ESI) m/z: 1040.6 [M + H] ⁺ R _f (60 % EtOAc in heptane) = 0.33  ((1S,4r)-4-(((S)-1-(((S)-5-carboxy-5-(3-((S)-1,3-dicarboxypr opyl)ureido)pentyl)amino)-3-(4- iodophenyl)-1-oxopropan-2-yl)carbamoyl)cyclohexyl)methanamin ium trifluoroacetate  

((1S,4r)-4-(((S)-1-(((S)-5-carboxy-5-(3-((S)-1,3-dicarboxypr opyl)ureido)pentyl)amino)-3-(4- iodophenyl)-1-oxopropan-2-yl)carbamoyl)cyclohexyl)methanamin ium trifluoroacetate was obtained from di-tert-butyl (((S)-1-(tert-butoxy)-6-((S)-2-((1r,4S)-4-(((tert- butoxycarbonyl)amino)methyl)cyclohexane-1-carboxamido)-3-(4- iodophenyl)propanamido)-1- oxohexan-2-yl)carbamoyl)-L-glutamate, following general procedure IV, as a viscous oil (TFA salt – 248 mg, 0.29 mmol, 99%) ¹ H NMR (400 MHz, (CD ₃ ) ₂ SO) δ 7.96 – 7.85 (m, 2H), 7.74 – 7.65 (m, 4H), 7.60 (d, J = 7.9 Hz, 2H), 7.02 (d, J = 7.9 Hz, 2H), 6.31 (t, J = 9.6 Hz, 2H), 4.47 – 4.36 (m, 1H), 4.14 – 3.98 (m, 3H), 3.07 – 2.93 (m, 3H), 2.92 – 2.84 (m, 1H), 2.76 – 2.59 (m, 3H), 2.34 – 2.16 (m, 2H), 2.12 – 2.00 (m, 1H), 1.98 – 1.85 (m, 1H), 1.81 – 1.61 (m, 5H), 1.57 – 1.42 (m, 1H), 1.41 – 1.20 (m, 6H), 1.17 – 1.00 (m, 1H), 0.98 – 0.81 (m, 2H). ¹³ C NMR (101 MHz, (CD ₃ ) ₂ SO) δ 174.6, 174.5, 174.1, 173.7, 170.8, 157.3, 137.9, 136.6, 131.7, 91.9, 53.3, 52.3, 51.7, 44.3, 43.2, 35.0, 31.7, 29.9, 28.9, 28.8, 28.7, 28.3, 28.0, 27.5, 22.6. MS (ESI) m/z: 732.4 [M - H] ⁺ ((1S,4r)-4-(((S)-1-(((S)-5-carboxy-5-(3-((S)-1,3-dicarboxypr opyl)ureido)pentyl)amino)-3-(3- iodophenyl)-1-oxopropan-2-yl)carbamoyl)cyclohexyl)methanamin ium trifluoroacetate ((1S,4r)-4-(((S)-1-(((S)-5-carboxy-5-(3-((S)-1,3-dicarboxypr opyl)ureido)pentyl)amino)-3-(3- iodophenyl)-1-oxopropan-2-yl)carbamoyl)cyclohexyl)methanamin ium trifluoroacetate was obtained from di-tert-butyl (((S)-1-(tert-butoxy)-6-((S)-2-((1r,4S)-4-(((tert- butoxycarbonyl)amino)methyl)cyclohexane-1-carboxamido)-3-(3- iodophenyl)propanamido)-1-   oxohexan-2-yl)carbamoyl)-L-glutamate, following general procedure IV, as a transparent oil (TFA salt –116 mg, 0.14 mmol, 94%) ¹ H NMR (400 MHz, D ₂ O) δ 7.63 – 7.53 (m, 2H), 7.22 (d, J = 7.7 Hz, 1H), 7.08 (t, J = 7.8 Hz, 1H), 4.46 (q, J = 7.8 Hz, 1H), 4.26 (dd, J = 8.9, 5.2 Hz, 1H), 4.22 – 4.08 (m, 1H), 3.17 (q, J = 6.3 Hz, 2H), 3.05 – 2.89 (m, 3H), 2.89 – 2.78 (m, 3H), 2.49 (q, J = 7.5 Hz, 3H), 2.27 – 2.08 (m, 2H), 2.02 – 1.90 (m, 1H), 1.91 – 1.77 (m, 5H), 1.77 – 1.70 (m, 2H), 1.66 – 1.58 (m, 2H), 1.45 – 1.27 (m, 5H), 1.27 – 1.13 (m, 2H), 1.12 – 0.94 (m, 3H). MS (ESI) m/z: 732.4 [M - H] ⁺ ((1R,4r)-4-(((R)-1-(((S)-5-carboxy-5-(3-((S)-1,3-dicarboxypr opyl)ureido)pentyl)amino)-3-(5- iodo-1H-indol-3-yl)-1-oxopropan-2-yl)carbamoyl)cyclohexyl)me thanaminium trifluoroacetate ((1R,4r)-4-(((R)-1-(((S)-5-carboxy-5-(3-((S)-1,3-dicarboxypr opyl)ureido)pentyl)amino)-3-(5-iodo- 1H-indol-3-yl)-1-oxopropan-2-yl)carbamoyl)cyclohexyl)methana minium trifluoroacetate was obtained from di-tert-butyl (((S)-1-(tert-butoxy)-6-((R)-2-((1r,4R)-4-(((tert- butoxycarbonyl)amino)methyl)cyclohexane-1-carboxamido)-3-(5- iodo-1H-indol-3- yl)propanamido)-1-oxohexan-2-yl)carbamoyl)-L-glutamate, following general procedure IV. In this case the deprotection cocktail was 1/1 TFA:TIPS:phenol (95:5:5)/DCM. The deprotected compound was purified by preparatory HPLC to obtain the title compound as a transparent oil (TFA salt –23 mg, 0.03 mmol, 34%) ¹ H NMR (600 MHz, CD3OD-d4) δ 7.93 (s, 1H), 7.33 (d, J = 8.2 Hz, 1H), 7.16 (d, J = 8.3 Hz, 1H), 7.09 (d, J = 5.8 Hz, 1H), 4.58 – 4.52 (m, 1H), 4.37 – 4.28 (m, 1H), 4.23 – 4.15 (m, 1H), 3.21 – 3.13 (m, 2H), 3.12 – 2.99 (m, 2H), 2.83 – 2.73 (m, 2H), 2.48 – 2.34 (m, 2H), 2.28 – 2.18 (m, 1H), 2.16 – 2.07 (m, 1H), 1.87 (q, J = 11.6, 9.3 Hz, 5H), 1.78 – 1.66 (m, 2H), 1.65 – 1.51 (m, 2H), 1.48 – 1.30 (m, 3H), 1.30 – 1.15 (m, 2H), 1.13 – 0.99 (m, 2H). ¹³ C NMR (151 MHz, CD3OD) δ 178.3, 175.1, 174.8, 174.6, 174.6, 160.0, 137.0, 131.6, 130.7, 128.5, 125.9, 114.5, 110.5, 82.8, 55.8, 54.2, 53.5, 46.3, 45.4, 40.0, 36.7, 32.7, 30.9, 30.3, 29.6, 29.4, 29.1, 28.5, 23.9. (((S)-1-carboxy-5-((S)-2-((1S,4S)-4-(((S)-3-(4-iodophenyl)-2 -(2-(4,7,10-tris(carboxymethyl)- 1,4,7,10-tetraazacyclododecan-1-yl)acetamido)propanamido)met hyl)cyclohexane-1- carboxamido)-3-(naphthalen-2-yl)propanamido)pentyl)carbamoyl )-L-glutamic acid trifluoroacetic acid salt (Im)  

The title compound was obtained from di-tert-butyl (((S)-6-((S)-2-((1S,4S)-4-(((S)-2-amino-3-(4- iodophenyl)propanamido)methyl)cyclohexane-1-carboxamido)-3-( naphthalen-2- yl)propanamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L -glutamate following general procedure V – A. Preparatory HPLC purification was carried out using a gradient of 0% to 100% of B over 15 minutes. Fractions containing the desired compound were lyophilised to obtain Im as white powder. (11 mg, 0.01 mmol, 13%). 1H NMR (600 MHz, CD3CN + 10% D2O) δ 7.88 – 7.76 (m, 3H), 7.72 – 7.55 (m, 3H), 7.52 – 7.41 (m, 1H), 7.41 – 7.34 (m, 1H), 7.07 – 6.96 (m, 3H), 4.60 – 4.52 (m, 1H), 4.52 – 4.42 (m, 1H), 4.25 – 4.16 (m, 1H), 4.12 – 4.01 (m, 1H), 3.57 (s, 8H), 3.29 – 2.70 (m, 18H), 2.42 – 2.31 (m, 2H), 2.11 – 1.99 (m, 1H), 1.87 – 1.73 (m, 1H), 1.72 – 1.57 (m, 2H), 1.56 – 1.41 (m, 2H), 1.40 – 1.27 (m, 2H), 1.27 – 1.15 (m, 3H), 1.15 – 1.03 (m, 1H), 0.78 – 0.61 (m, 4H). 1 ³ C NMR (151 MHz, CD ₃ CN + 10% D ₂ O) δ 178.6, 178.51, 176.52, 176.4, 176.2, 175.9, 173.10, 173.07, 172.2, 161.6 (q, J = 34.6 Hz), 159.4, 138.5, 138.5, 137.9, 135.9, 134.4, 133.3, 132.80, 132.76, 128.9, 128.8, 128.57, 128.53, 127.2, 126.7, 117.7 (overlapped with CD3CN signal, q, J = 292.9 Hz) 92.6, 55.8, 55.5, 53.8, 53.3, 46.3, 46.1, 45.4, 43.7, 40.8, 39.5, 39.4, 38.6, 38.3, 37.7, 37.5, 31.9, 30.9, 30.3, 30.2, 30.1, 29.71, 29.66, 29.60, 29.5, 29.1, 27.9, 27.6, 25.2, 23.3, 23.1. (((S)-1-carboxy-5-((S)-2-((S)-3-(4-iodophenyl)-2-(2-(4,7,10- tris(carboxymethyl)-1,4,7,10- tetraazacyclododecan-1-yl)acetamido)propanamido)-3-(naphthal en-2- yl)propanamido)pentyl)carbamoyl)-L-glutamic acid trifluoroacetic acid salt (In)  

The title compound was obtained from di-tert-butyl (((S)-6-((S)-2-((S)-2-amino-3-(4- iodophenyl)propanamido)-3-(naphthalen-2-yl)propanamido)-1-(t ert-butoxy)-1-oxohexan-2- yl)carbamoyl)-L-glutamate following general procedure V – A. Preparatory HPLC purification was carried out using a gradient of 0% to 100% of B over 15 minutes. Fractions containing the desired compound were lyophilised to obtain In as white powder. (10 mg, 0.01 mmol, 10%). ¹ H NMR (600 MHz, CD ₃ CN + 10% D ₂ O) δ 7.83 (q, J = 8.3 Hz, 3H), 7.68 (s, 1H), 7.52 (d, J = 7.8 Hz, 2H), 7.46 (q, J = 7.5 Hz, 2H), 7.36 (dd, J = 8.6, 1.8 Hz, 1H), 6.85 (d, J = 7.9 Hz, 2H), 4.61 (t, J = 7.5 Hz, 1H), 4.58 – 4.49 (m, 1H), 4.20 (dd, 1H), 4.06 (dd, J = 8.7, 4.9 Hz, 1H), 3.85 – 3.42 (m, 6H), 3.31 (s, 23H), 3.24 – 3.08 (m, 1H), 3.08 – 2.83 (m, 2H), 2.67 (dd, J = 13.8, 9.4 Hz, 1H), 2.36 (td, J = 7.5, 2.3 Hz, 2H), 2.09 – 2.01 (m, 1H), 1.88 – 1.77 (m, 1H), 1.64 – 1.53 (m, 1H), 1.53 – 1.42 (m, 1H), 1.33 – 1.18 (m, 2H), 1.18 – 1.04 (m, 2H). ¹³ C NMR (151 MHz, CD3CN + 10% D2O) δ 176.34, 176.18, 175.85, 172.69, 172.06, 161.74, 161.51, 161.28, 161.05, 159.37, 138.51, 138.33, 138.17, 135.55, 134.41, 133.39, 132.76, 129.03, 128.97, 128.79, 128.64, 128.60, 127.27, 126.83, 92.43, 55.64, 55.36, 53.74, 53.33, 39.70, 38.86, 37.62, 32.02, 30.92, 28.94, 27.98, 26.69, 26.16, 24.81, 23.20. (((S)-1-carboxy-5-((S)-3-(2-iodophenyl)-2-((1r,4S)-4-((2-(4, 7,10-tris(carboxymethyl)-1,4,7,10- tetraazacyclododecan-1-yl)acetamido)methyl)cyclohexane-1- carboxamido)propanamido)pentyl)carbamoyl)-L-glutamic acid trifluoroacetic acid salt (Ii)  

The title compound was obtained from di-tert-butyl (((S)-6-((S)-2-((1r,4S)-4- (aminomethyl)cyclohexane-1-carboxamido)-3-(2-iodophenyl)prop anamido)-1-(tert-butoxy)-1- oxohexan-2-yl)carbamoyl)-L-glutamate following general procedure V – A. Preparatory HPLC purification was carried out using a gradient of 0% to 100% of B over 15 minutes. Fractions containing the desired compound were lyophilised to obtain Ii as white powder. (22 mg, 0.02 mmol, 27%) 1H NMR (600 MHz, CD3CN + 10% D2O) δ 7.83 (dt, J = 7.7, 1.5 Hz, 1H), 7.32 – 7.26 (m, 1H), 7.26 – 7.20 (m, 1H), 6.95 (tt, J = 7.4, 1.9 Hz, 1H), 4.55 – 4.49 (m, 1H), 4.23 – 4.17 (m, 1H), 4.11 – 4.06 (m, 1H), 3.72 (d, J = 90.6 Hz, 5H), 3.29 – 2.79 (m, 13H), 2.39 – 2.29 (m, 3H), 2.13 – 2.01 (m, 1H), 1.88 – 1.76 (m, 1H), 1.76 – 1.66 (m, 3H), 1.66 – 1.59 (m, 1H), 1.59 – 1.47 (m, 1H), 1.43 – 1.29 (m, 2H), 1.29 – 1.12 (m, 4H), 0.93 – 0.78 (m, 3H). 1 ³ C NMR (151 MHz, CD3CN + 10% D2O) δ 178.5, 178.4, 176.46, 176.43, 176.3, 175.82, 175.79, 172.54, 172.51, 161.46 (q, J = 34.7 Hz), 159.4, 140.8, 140.6, 131.9, 129.8, 129.5, 117.7 (overlapped with CD ₃ CN signal, q, J = 292.9 Hz), 101.4, 62.8, 55.9, 54.2, 54.1, 53.96, 53.93, 53.32, 53.28, 46.4, 45.4, 42.9, 39.6, 39.4, 37.7, 32.1, 32.0, 30.9, 30.34, 30.29, 29.61, 29.58, 29.5, 29.2, 29.1, 28.1, 27.9, 23.4, 23.2. (((S)-1-carboxy-5-((S)-3-(3-iodophenyl)-2-((1r,4S)-4-((2-(4, 7,10-tris(carboxymethyl)-1,4,7,10- tetraazacyclododecan-1-yl)acetamido)methyl)cyclohexane-1- carboxamido)propanamido)pentyl)carbamoyl)-L-glutamic acid trifluoroacetic acid salt (Ij)  

The title compound was obtained from ((1S,4r)-4-(((S)-1-(((S)-5-carboxy-5-(3-((S)-1,3- dicarboxypropyl)ureido)pentyl)amino)-3-(3-iodophenyl)-1-oxop ropan-2- yl)carbamoyl)cyclohexyl)methanaminium trifluoroacetate following general procedure V – B. Preparatory HPLC purification was carried out using a method consisting of 8 min of 100% A after injection followed by a gradient from 0 to 100% B over 20 min. Fractions containing the desired compound were lyophilised to obtain Ij as white powder. (24 mg, 0.02 mmol, 30%) 1H NMR (600 MHz, CD3CN + 10% D2O) δ 7.59 – 7.54 (m, 2H), 7.22 (ddt, J = 7.8, 2.9, 1.3 Hz, 1H), 7.08 – 7.02 (m, 1H), 4.45 – 4.38 (m, 1H), 4.22 – 4.17 (m, 1H), 4.14 – 4.07 (m, 1H), 3.89 – 3.54 (m, 5H), 3.37 – 2.89 (m, 20H), 2.79 (dd, J = 13.8, 9.0 Hz, 1H), 2.37 (ddt, J = 8.2, 6.5, 1.6 Hz, 2H), 2.12 – 2.01 (m, 2H), 1.89 – 1.77 (m, 1H), 1.76 – 1.66 (m, 4H), 1.64 – 1.53 (m, 2H), 1.44 – 1.32 (m, 3H), 1.32 – 1.17 (m, 4H), 0.96 – 0.84 (m, 2H). 1 ³ C NMR (151 MHz, CD ₃ CN + 10% D ₂ O) δ 178.6, 178.5, 176.50, 176.48, 176.3, 175.88, 175.86, 172.73, 172.70, 161.6 (q, J = 34.5 Hz), 159.4, 141.0, 139.2, 136.7, 131.4, 129.8, 117.7 (overlapped with CD3CN signal, q, J = 293.2 Hz), 94.7, 55.9, 55.3, 55.2, 53.93, 53.88, 53.31, 53.28, 46.4, 45.39, 45.37, 39.6, 39.4, 37.9, 37.7, 32.1, 32.0, 30.4, 30.3, 29.79, 29.77, 29.4, 29.2, 29.1, 27.98, 27.94, 23.4, 23.2. (((S)-1-carboxy-5-((S)-3-(4-iodophenyl)-2-((1r,4S)-4-((2-(4, 7,10-tris(carboxymethyl)-1,4,7,10- tetraazacyclododecan-1-yl)acetamido)methyl)cyclohexane-1- carboxamido)propanamido)pentyl)carbamoyl)-L-glutamic acid trifluoroacetic acid salt (Ik)  

The title compound was obtained from ((1S,4r)-4-(((S)-1-(((S)-5-carboxy-5-(3-((S)-1,3- dicarboxypropyl)ureido)pentyl)amino)-3-(4-iodophenyl)-1-oxop ropan-2 yl)carbamoyl)cyclohexyl)methanaminium trifluoroacetate following general procedure V – B. Preparatory HPLC purification was carried out using a method consisting of 8 min of 100% A after injection followed by a gradient from 0 to 100% B over 20 min. Fractions containing the desired compound were lyophilised to obtain Ik as white powder. (42 mg, 0.04 mmol, 53%) ¹ H NMR (600 MHz, CD ₃ CN + 10% D ₂ O) δ 7.60 (d, 2H), 6.98 (d, 2H), 4.43 – 4.34 (m, 1H), 4.20 – 4.11 (m, 1H), 4.11 – 4.00 (m, 1H), 3.71 – 3.39 (m, 8H), 3.31 – 2.98 (m, 16H), 2.98 – 2.85 (m, 2H), 2.78 (dd, J = 13.5, 8.6 Hz, 1H), 2.39 – 2.28 (m, 2H), 2.10 – 1.97 (m, 2H), 1.87 – 1.74 (m, 1H), 1.73 – 1.61 (m, 4H), 1.60 – 1.44 (m, 3H), 1.41 – 1.27 (m, 1H), 1.27 – 1.09 (m, 4H), 0.92 – 0.74 (m, 3H). ¹³ C NMR (151 MHz, CD ₃ CN + 10% D ₂ O) δ 178.8, 176.8, 176.7, 176.1, 172.9, 162.1 (q, J = 34.2), 159.5, 138.3, 138.1, 132.6, 118.8 (overlapped with CD ₃ CN signal, q, J = 293 Hz), 92.5, 55.8, 55.3, 53.9, 53.3, 46.2, 45.3, 39.6, 37.8, 37.6, 32.0, 30.9, 30.3, 30.2, 29.6, 29.3, 29.1, 27.8, 23.3. (((S)-1-carboxy-5-((R)-3-(5-iodo-1H-indol-3-yl)-2-((1r,4R)-4 -((2-(4,7,10-tris(carboxymethyl)- 1,4,7,10-tetraazacyclododecan-1-yl)acetamido)methyl)cyclohex ane-1- carboxamido)propanamido)pentyl)carbamoyl)-L-glutamic acid trifluoroacetic acid salt (Il)  

The title compound was obtained from ((1R,4r)-4-(((R)-1-(((S)-5-carboxy-5-(3-((S)-1,3- dicarboxypropyl)ureido)pentyl)amino)-3-(5-iodo-1H-indol-3-yl )-1-oxopropan-2- yl)carbamoyl)cyclohexyl)methanaminium trifluoroacetate following general procedure V – B. Preparatory HPLC purification was carried out using a method consisting of 8 min of 100% A after injection followed by a gradient from 0 to 100% B over 20 min. Fractions containing the desired compound were lyophilised to obtain Il as white powder. (14 mg, 0.01 mmol, 46%) 1H NMR (600 MHz, CD ₃ CN + 10% D ₂ O) δ 7.91 (s, 1H), 7.36 (dd, J = 8.5, 1.7 Hz, 1H), 7.21 (d, J = 8.5 Hz, 1H), 7.08 (d, J = 8.3 Hz, 1H), 4.44 (t, J = 7.1 Hz, 1H), 4.21 (dt, J = 8.9, 4.4 Hz, 1H), 4.07 (dt, J = 8.9, 4.9 Hz, 1H), 3.90 – 3.50 (m, 8H), 3.30 – 2.96 (m, 18H), 2.37 (td, J = 7.0, 6.1, 2.9 Hz, 2H), 2.13 – 2.02 (m, 3H), 1.89 – 1.79 (m, 1H), 1.77 – 1.61 (m, 5H), 1.52 (dtd, J = 14.0, 9.2, 5.3 Hz, 1H), 1.46 – 1.36 (m, 1H), 1.35 – 1.21 (m, 5H), 1.20 – 1.11 (m, 2H), 0.96 – 0.82 (m, 3H). 1 ³ C NMR (151 MHz, CD3CN + 10% D2O) δ 178.3, 176.4, 176.2, 175.8, 173.2, 161.4 (q, J = 34.7 Hz), 159.5, 136.3, 131.2, 130.5, 128.4, 126.0, 116.7 (overlapped with CD ₃ CN signal, q, J = 292 Hz), 114.8, 110.3, 82.8, 56.0, 55.1, 53.9, 53.3, 46.4, 45.4, 39.6, 39.4, 37.8, 32.2, 32.0, 30.9, 30.4, 30.3, 29.5, 29.1, 29.0, 28.4, 28.0, 27.9, 23.4.   Example 4: synthesis of PSMA‐targeting radiopharmaceu tical precursor:  di-tert-butyl (((S)-1-(tert-butoxy)-1-oxo-6-((S)-3-(4-(trimethylstannyl)ph enyl)-2-((1r,4S)-4- ((2-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraa zacyclododecan-1- yl)acetamido)methyl)cyclohexane-1-carboxamido)propanamido)he xan-2-yl)carbamoyl)-L- glutamate  

To an LuG-linker solution (1 eq.) in DCM, Et3N (6 eq.), DOTA-mono-NHS-tris(tBu-ester) (1.2 eq.) were added and stirred overnight (~16 hours). Afterwards the reaction mixture was evaporated under reduced pressure. One microwave vial was charged with Pd(OAc) ₂ (1.5 mg, 0.007 mmol) and the crude LuG-linker DOTA-functionalized mixture (7.7 mg, 0.26 mmol). The vial was sealed and purged. Then dry/degassed THF (400 uL) was added to the vial. In another MW vial, hexamethylditin (20.5 uL, 0.099 mmol) was added and the vial purged, thereafter dry/degassed THF was added (300 uL). The hexamehtylditin solution was then added to the Pd(OAc)2 and meCgPPH mixture and the solution stirred for 5 minutes at room temperature. Thereafter, fully tBu-protected 4 (48.0 mg, 0.033 mmol) which was previously weighed in a MW vial and dissolved in dry/degassed THF (300 uL) was added to the Pd/meCgPPH/Sn mixture. The vial was then placed on a MW system and heated to 70 °C for 30 min. The mixture was dried under a stream of air and re-dissolved in 4 mL 60:40:0.1 (MeCN:H2O:TFA). The mixture was filtered over a prep HPLC filter and injected into the prepHPLC system in a gradient of 60 to 100% B. which yielded the desired product as a white solid. (12.5 mg, 0.0084 mmol, 25%). MS (ESI) m/z: 747.47 [M + 2H] ²⁺ Example 5: Radioastatination and radiochemical conversi on (RCC)  Compound Ib was provided in a two‐step labelling p rocedure:   

The stannane precursor was added to a solution of c hloramine‐T, methanol,  ²¹¹ At and acetic  acid. The mixture was stirred for 30 minutes at roo m temperature (step 1), followed by drying  under a nitrogen stream. The radioastatinated product� �was deprotected by addition of  trifluoroacetic acid (TFA) and heating to 60 °C for  30 minutes (step 2). The radiochemical  conversion (%RCC) for this procedure is shown in Tab le 1:  Table 1:     Thus, the PSMA analogue, provided in a two‐step la beling procedure, resulted in a RCC of  >60%, which is surprising, since a similar labelli ng procedure, reported in WO2019/157037  yielded a labelled PSMA analogue ([ ²¹¹ At]VK‐02‐90) in 12.5% radiochemical yield (RC Y, non‐ decay corrected), over two steps. No detailed data i s provided for the method used in  WO2019/157037 (one pot procedure, 3 modification), how ever an RCY of maximum 26% was  reported.  RCC as stated above refers to radiochemical conversio n, and is used as a measure of how much  of the added activity that is converted to the desi red product, as demonstrated by  chromatography, typically radio‐TLC or radio‐HPLC.  RCC measurements are made before a  potential work‐up or purification is carried out. I n this way, RCC can be said to measure the  efficiency of the chemical radiolabeling reaction. RCY  refers to radiochemical yield, and is used  as a measure of how much of the added activity end s up as the desired product in a purified  form, typically with an associated radiochemical purit y (RCP) that says how much of the  activity in the purified product is present as the  actual desired product. In this sense, RCY  reflects both the efficiency of the labeling (RCC) a nd the efficiency of the work‐up procedure,   

since product may be lost during purification. How ever, unless a very inefficient type of  purification is used, RCY will be similar to RCC, w ith RCC being slightly higher. In  WO2019/157037, purification was done by HPLC, a state ‐of‐the‐art, standard procedure.  Accordingly, RCY and RCC would be expected to be si milar, typically with a difference between  the two of about 5‐15%. In this sense, the differ ence between a reported RCY of 26% and an  RCC of 71% is substantial and reflects a difference� �in the efficiency of the radiolabeling  reactions themselves. It should be noted that efficie nt radiochemistry is crucial for commercial  use as it limits loss of the radionuclide, makes pu rification easier, limits radioactive waste, and  limits exposure of personnel to radiation.  Example 6: 68Ga‐labeling  ⁶⁸ Ga [E _β+,max = 1.9 MeV (88%), t _1/2  = 68 min] was eluted from a  ⁶⁸ Ge (t _1/2  = 271 d) generator (ITM  AG, Munich, Germany), based on silica gel modified w ith dodecyl gallate, as [ ⁶⁸ Ga]GaCl ₃  in 0.1  M HCl and trapped in an SCX cartridge (HyperSep, Th ermoFischer). The trapped [ ⁶⁸ Ga]Ga ³⁺  was  eluted with 300 µL of a 5 M NaCl/HCl solution gen erally giving 500‐600 MBq of activity and  employed in further radiolabellings.  Radiolabelling of the DOTA‐containing peptidomimetics� �was carried out as follows. 40 uL of  [ ⁶⁸ Ga]Ga ³⁺  eluate (∼ 50 – 80 MBq) were mixed with 40 µL of 1.0 M H EPES (4‐(2‐hydroxyethyl)‐ 1‐piperazineethanesulfonic acid, pH 4). If needed, t he pH of the solution was adjusted to 3.8‐ 4.2 by addition of 10% NaOH _(aq.) . Thereafter, 5 µl (internalization experiment)� �or 2 µl (PET  imaging) of a 1 mM solution of the DOTA‐bearing p eptide was added and the reaction mixture  was heated to 95 °C for 15 min (internalization ex periment) or  5 min (PET imaging),  respectively. Radiolabelling efficiency was determined  by RP‐HPLC (5‐95% B in 5 minutes ‐  Chromolith RP‐18e 100x4.6) and was deemed to be &g t;98% for all the radiolabelled compounds.   Example 7: in vitro test of binding affinity and in ternalisation  Compounds Ii, Ij, Ik, Il, Im and In was provided u sing the same method as disclosed in Example  3 and in vitro test of the binding affinity and in ternalisation of the compounds were examined  using the method as described in Benesova et al, 20 16 ⁹ . The iodine isotope used was  ¹²⁷ I.   For internalisation determinations, a day before the  experiment, PSMA(+) LNCaP cells were  seeded in a poly‐L‐lysine coated 24‐well plate  (10 ⁵  cells per well) and maintained at 37°C in a n  atmosphere of 5% CO ₂  under supplemented RMPI medium (10% Fetal Calf  Serum, 1% sodium  pyruvate, 1% FIS). Cells were incubated with 250 µL  of radiolabelled compound diluted in RPMI  medium (final concentration of [ ⁶⁸ Ga]‐peptide: 30 nM) and 500 μM of PMPA (2� �   phosphonomethyl‐pentanedioic acid) for the blocked se ries, for 45 min at 37°C. Cellular uptake  was interrupted by washing the cells with ice‐cold� �PBS (3 x 1 mL). Surface bound radioactivity  was removed by incubating twice with 0.5 mL glycine� �(50 mM, pH = 2.8) for 5 min. Thereafter,  cells were washed with PBS (1 mL) and lysed employi ng NaOH (0.3 M) during 10 min. Lysates  and surface‐bound activity were collected and measur ed in a gamma‐counter (Perkin Elmer  2480, Wizard, Gamma Counter). The cell uptake was ca lculated as percent of the initially  added radioactivity bound to 105 cells [%ID/105 cells ].  For internalisation studies androgen‐sensitive human  prostate adenocarcinoma cells (LNCaP)  highly overexpressing PSMA were incubated with the  ⁶⁸ Ga‐labeled compounds resulting in  specific cell surface binding of all tested compounds  (Table 2).   Table 2. Internalisation data*.  * Data are expressed as mean ± SD (n=3),  ^+ 68 Ga‐labeled compounds. Specific cell uptake was� � determined by blockage using 500 µM 2‐PMPA. Values  are expressed as % of applied  radioactivity (IA) bound to 10 ⁵  cells.  The results are shown in Table 2. All compounds rev ealed comparable internalisation  properties as PSMA‐617, except IN showing a reduced  internalised fraction. Especially  compound Ij and Im displayed a specific internalizati on higher than or comparable to PSMA‐ 617, which was surprising, as modifying this amino a cid residue in the linker region of PSMA  inhibitors can have significant effects on binding an d internalisation, as is reported in Benesova  et al., 2016 ⁹ . Internalisation is the key predictor for succ essful PSMA therapy.     

Example 8: in vivo evalution of the PSMA targetin g radioligands  Compounds Ii, Ij, Ik, Il and Im which showed intern alisation comparable to PSMA‐617 as shown  in Example 7 were selected for in vivo evaluation i n mice.   For the experimental tumor models 1×10 ⁷  cells of LNCaP (in 50% Matrigel; Becton Dickins on)  were subcutaneously inoculated into the right flank o f 7‐ to 8‐week‐old male BALB/c nu/nu  mice (Janvier). For imaging studies, mice were anesth etized (2% isoflurane) and 0.5 nmol of  the  ⁶⁸ Ga‐labeled compound in 0.9% NaCl (pH 7) were� �injected into the tail vein. PET imaging  was performed with µPET/MRI scanner (BioSpec 3T, Bru ker) with a dynamic scan for 60 min.  The images were iteratively reconstructed (MLEM 0.5 a lgorithm, 12 iterations) and were  converted to SUV images. Quantification was done usin g a ROI (region of interest) technique  and data in expressed in time activity curves as SU V _body weight . All animal experiments complied  with the current laws of the Federal Republic of Ge rmany.  PET imaging: tumor uptake and pharmacokinetic profile� � Figures 2 to 7 show the pharmacokinetic study with  small‐animal PET imaging. Time activity  curves for non‐target organs and tumor after inject ion of 0.5 nmol  ⁶⁸ Ga‐labeled compounds in  LNCaP‐ tumor‐bearing athymic nude mice (right trun k) up to 60 min p.i.. SUV=standardized  uptake value.   The pharmacokinetic properties and tumor targeting pro perties of the modified compounds  were found to be comparable or superior to the pare ntal reference PSMA‐617 (see Figures 2 ‐  7). For example and surprisingly, the tumor‐to‐mus cle ratio for Ii increased at later time points  compared to PSMA‐617 (Figure 3B). Im (Figure 7B) s howed a higher tumor uptake compared  to PSMA‐617 (Figure 2B), whereas a more reversible� �tumor accumulation could be seen for IK  (Figure 5B). Moreover, the total uptake in the inves tigated organs and tumor, as well as the  excretion profile, indicate the suitability of the ne w compounds as radiopharmaceuticals ‐ for  example as 211At labeled PSMA‐inhibitors.          

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